23 April 2012
Summary:
-Initial data collection and analysis of data for gill and gonadal tissues.

Procedure:
-Following lab protocol for data analysis, can be found under home page "how-to" section, titled "analyze qPCR data crude method"

Sample - Gill	Original Vtg	Converted Value	EFLA Original	EFLA Converted	Vtg/EFLA Values
CT1	0	2.71644E+11	21.7	79060.58072
CT2	0	2.71644E+11	30.77	146.5804329
CT3	0	2.71644E+11	27.44	1475.951205
CT4	0	2.71644E+11	26.08	3790.565452
EST1	0	2.71644E+11	24.77	9403.207072
EST2	0	2.71644E+11	28.46	727.5251313
EST3	0	2.71644E+11	28.13	914.6269627
EST4	0	2.71644E+11	30.68	156.0213387
Sample - Gill	Original ER2	Converted Value	EFLA Original	EFLA Converted	ER2/EFLA Values
CT1	34.97	7.962253492	21.7	79060.58072	0.000100711
CT2	0	2.71644E+11	30.77	146.5804329
CT3	39.18	0.429520602	27.44	1475.951205	0.000291013
CT4	0	2.71644E+11	26.08	3790.565452
EST1	37.1	1.817523354	24.77	9403.207072	0.000193288
EST2	0	2.71644E+11	28.46	727.5251313
EST3	0	2.71644E+11	28.13	914.6269627
EST4	0	2.71644E+11	30.68	156.0213387
Sample - Gonadal	Original Vtg	Converted Value	EFLA Original	EFLA Converted	Vtg/EFLA Values
CT1	0	2.71644E+11	26.89	2161.384337
CT2	0	2.71644E+11	29.32	400.6969161
CT3	39.39	0.371306028	25.93	4206.142674	8.82771E-05
CT4	0	2.71644E+11	21.8	73763.24262
EST1	33.09	29.32864508	26.55	2736.150615	0.010718944
EST2	0	2.71644E+11	29.73	301.5254679
EST3	33.53	21.615436	24.25	13486.52308	0.001602743
EST4	0	2.71644E+11	25.41	6032.648204
Sample - Gonadal	Original ER2	Converted Value	EFLA Original	EFLA Converted	ER2/EFLA Values
CT1	36.01	3.87068652	26.89	2161.384337	0.001790837
CT2	38.41	0.73266931	29.32	400.6969161	0.001828488
CT3	37.87	1.065506246	25.93	4206.142674	0.000253321
CT4	36.43	2.892570815	21.8	73763.24262	3.92143E-05
EST1	36.19	3.416425856	26.55	2736.150615	0.001248625
EST2	36.5	2.755497365	29.73	301.5254679	0.009138523
EST3	36.49	2.774674226	24.25	13486.52308	0.000205737
EST4	37.57	1.311946122	25.41	6032.648204	0.000217474

Gill Tissue cq Values

Sample	VITELLOGENIN	ESTROGEN RECEPTOR	GILL TISSUE EFLA cq VALUES
CT1	NA	34.97	21.70
CT2	NA	NA	30.77
CT3	NA	39.18	27.44
CT4	NA	NA	26.08
EST1	NA	37.10	24.77
EST2	NA	NA	28.46
EST3	NA	NA	28.13
EST4	NA	NA	30.68

Gonadal Tissue cq Values

Sample	VITELLOGENIN	ESTROGEN RECEPTOR	GONADAL TISSUE EFLA cq VALUES
CT1	NA	36.01	26.89
CT2	NA	38.41	29.32
CT3	39.39	37.87	25.93
CT4	NA	36.43	21.80
EST1	33.09	36.19	26.55
EST2	NA	36.50	29.73
EST3	33.53	36.49	24.25
EST4	NA	37.57	25.41

---

20 April 2012
Summary:
-Ran gonadal tissue with EFLA elongation factor
-Start analysis

Procedure:
-qPCR was done to lab protocol
-Master mix was same quantities as on 21 March 2012
-Only difference was the primer was EFLA

Well Placement

well	5	6
1	CT1	EFLA POSITIVE CONTROL
2	CT2	NO TEMPLATE
3	CT3	NO TEMPLATE
4	CT4	BLANK
5	EST1	""
6	EST2	""
7	EST3	""
8	EST4	""

Amplification Curve - Elongation factor with gonadal tissue


Melting Curve - The two lower curves between 3000-3500RFU are the no template controls.


Melting Peak - The adnormal melting peaks below 200 are the no template controls


Next Step:
-Analysis of cq data between EFLA primers and genes of interest.

---

17 April 2012
Summary:
-Converted CT 1-4, EST 1-4 gonadal RNA tissue to cDNA.
-Ran qPCR on gonadal tissues

Procedure:
-For cDNA conversion lab protocol was followed

SPEC OF GONADAL TISSUE

SAMPLE	ng/ul
CT1	55.61
2	74.9
3	74.5
4	85.9
EST 1	153.97
2	85.6
3	86.0
4	146.1

Sample	RNA USED (ul)	H2O USED (ul)
CT1	17.75	0
CT2	13.18	4.57
CT3	13.25	4.5
CT4	11.49	6.26
EST1	6.41	11.34
EST2	11.53	6.22
EST3	11.47	6.28
EST4	6.76	10.99

-For qPCR procedure was done to lab protocol
-Gonadal tissue with 2 primers, vitellogenin and estrogen receptor, same as 05 April 2012
-Master mix same as 21 March 2012

Amplification Curve

Vitellogenin Amplification Curve

-At 30 cycles EST1 & 3 are shown, the positive control and CT3 are at roughly 36 cycles.

Melting Peak


ER2 Melting Curve

Vitellogenin Melting Curve


Well Placement
-Column 1: Vitellogenin primers
-Column 2: Positive controls and No templates
-Column 3: ER2 Primers

Well	1	2	3
A	CT1	Vg Pos. Con.	CT1
B	CT2	Vg No Temp.	CT2
C	CT3	Vg No Temp.	CT3
D	CT4	Blank	CT4
E	EST1	Blank	EST1
F	EST2	ER2 Pos. Con.	EST2
G	EST3	ER2 No Temp.	EST3
H	EST4	ER2 No Temp.	EST4

---

05 April 2012
Summary:
-Running qPCR for gill tissues using both Vg and ER2 primers.
-Vg samples used 2ul of templates versus only 1 ul of template in ER2 samples

Procedure:
-Master mix same as on 21 March 2012, only difference is Vg samples 2ul of template were used.

Well Placement:

Well	3 (All Vg Primers)	4	5 (All ER2 Primers)
1	CT1	Vg Positive Control	CT1
2	CT2	Vg - No template	CT2
3	CT3	Vg - No template	CT3
4	CT4		CT4
5	EST1		EST1
6	EST2	ER2 Positive Control	EST2
7	EST3	ER2 - No template	EST3
8	EST4	ER2 - No template	EST4

Amplification Curve


Melting Peaks


Next Step:
-Run Gonadal Tissue

---

21 March 2012
Summary:
-Running elongation factor with gill tissues x8 and positive control

Procedure:
-qPCR was done to lab protocol

Master mix - Total reaction 20 ul
So fast 10ul each x12 = 120ul
forward .5ul x12 = 6ul
Reverse .5ul x12 = 6 ul
H2O 8ul x12 = 96ul

Well Placement - All Gill Tissue
-Each (CT1, CT2) indicates the gill tissue from one oyster, CT = Control, EST = exposed

Well	1	2
1	CT1	POSITIVE CONTROL
2	CT2	NO TEMPLATE
3	CT3	NO TEMPLATE
4	CT4
5	EST 1
6	EST 2
7	EST 3
8	EST 4

Amplification Graph


Melting Graph



Next Step:
-Run ER qPCR

---

13 March 2012
Summary:
-Ran qPCR for Gill tissues with vitellogenin primer 1346/1345 (SRID)
-Total 11 samples, 8 gill tissue, 1 Vg control and x2 no templates

Procedure:
-Procedure was done to lab protocol.
-For clarification (CT1) is the name assignment to oyster control number 1, for each oyster like CT1 I took x4 tissue samples, this qPCR only is testing GILL tissue from the x8 oysters in my experiment. CT refers to controls and EST refers to estrogen treated.

Amplification Graph

Fig 1: The 1st line is amplification of the vitellogenin primer positive control.

Melting Curve


Table: Well Placement, Gill tissue

Well	6	7
1	CT1	VG POS. CONTROL
2	CT2	NO TEMPLATE
3	CT3	NO TEMPLATE
4	CT4
5	EST1
6	EST2
7	EST3
8	EST4

Next Step:
-Run elongation factor to test validity of cDNA with gill tissues

---

12 March 2012
Summary:
-Ran qPCR with GIll tissue cDNA
-total of 14 samples, 8 gill tissue, 1 vitellogenin control, 1 estrogen control, x2 no templates for each set of primers
-qPCR for vitellogenin showed no amplification, either tissues or control.
-qPCR for estrogen receptor gene showed good positive result.

Procedure:
-qPCR procedure was done to lab protocol
Table: well placement

Well	1	2
1	CT1	Vg Control
2	CT2	NO TEMP.
3	CT3	NO TEMP.
4	CT4
5	EST1
6	EST2	ER CONTROL
7	EST3	NO TEMP.
8	EST4	NO TEMP.

AMPLIFICATION RESULTS:

Fig 1: The 1st peak is amplification of the ER primers and the second is contamination from the first ER no template.

Melting Curve:

Fig 2: The first peak (to 420) is contamination of the 1st no template control, The second peak is the positive control
for the estrogen receptor primers in combination with pooled cDNA.

Next Step:
-Rerun vitellogenin primer with gill tissue
-Next run tissues with ER primer

---

07 March 2012
Summary:
-Only running one set of tissues, GILL for all 8 oysters sampled.
-Converted DNAsed RNA to cDNA for Gill tissues samples.

Procedure:
-Procedure was done to protocol.

-Spec of Gill samples and conversion to 1ug used in cDNA

Gill Tissue	ng/ul	260/280	260/230	RNA used (ul)	H2O used (ul)
CT1	621.59	1.99	1.58	1.61	16.14
CT2	73.50	2.01	1.20	13.61	4.14
CT3	71.03	1.99	1.39	14.08	3.67
CT4	100.2	2.01	1.24	9.98	7.77
EST1	100.6	2.01	1.46	9.94	7.80
EST2	78.5	1.95	1.06	12.74	5.01
EST3	81.2	2.00	1.32	12.31	5.44
EST4	68.5	1.96	0.88	14.59	3.15

Next Step:
-Run qPCR on cDNA samples for GIll tissue

---

06 March 2012
Summary:
-Ran DNAse procedure for samples CT2-4 and EST 2-4

Procedure:
-Procedure was done to commerical protocol from Roberts lab, I used the rigourous treatment due to the large amount of RNA in each sample.
-Each .5ml tube contains 5ug of RNA per 50ul of solution.

Sample	RNA ul	DEPC H2O ul
CT2 GILL	3.93	46.07
MANTLE	6.71	43.29
MUSCLE	7.59	42.41
GONAD	4.13	45.87
CT3 GILL	8.07	41.93
MANTLE	14.63	35.37
MUSCLE	44.74	5.26
GONAD	14.83	35.17
CT4 GILL	11.07	38.93
MANTLE	8.53	41.47
MUSCLE	32.06	17.94
GONAD	10.92	39.08
EST2 GILL	8.65	41.35
MANTLE	8.54	41.46
MUSCLE	26.89	23.11
GONAD	3.87	46.12
EST3 GILL	4.54	45.46
MANTLE	8.39	41.61
MUSCLE	9.01	41.00
GONAD	5.83	44.17
EST4 GILL	6.97	43.03
MANTLE	19.47	30.52
MUSCLE	7.32	42.68
GONAD	8.81	41.19

Next Step:
-Spec samples that I will convert to cDNA and run qPCR
-Run qPCR

---

05 March 2012
Summary:
-RNA qPCR analysis for 8 samples as listed on 01 March 2012.

RNA Amplification Graph - All samples showed negative for genomic material except the positive control (exponential curve) and CT1 Muscle (only slight product at the 38th cycle.


RNA Melting Curve - Positive control peak shows up well on melting point graph and CT1 Muscle "hump" can be seen between 82 and 85C. The product was amplified so late in the cycles (38) that it shouldn't be of concern.


Next Step:
-Treat last 24 samples with DNAse and prepare to run the on Wednesday 07 March 2012 through qPCR.
-By 09 March 2012, like to convert all RNA to cDNA and run qPCR.

---

01 March 2012
Summary:
-Ran spec on samples
-Ran qPCR on RNA samples
-made 5ul samples with a concentration of 40ng per ul

Procedure:
-Amount of RNA used

Sample	RNA(ul)	H2O(ul)
CT1 GILL	0.463	4.54
MANTLE	0.896	4.10
MUSCLE	2.52	2.48
GONAD	2.65	2.35
EST1 GILL	2.46	2.54
MANTLE	2.42	2.57
MUSCLE	2.19	2.81
GONAD	2.35	2.65

-Make sure to use DEPC water for samples containing RNA.
-example
ex 1ug/25 = .04 or 40 ng
setup c1v1=c2v2
(431.83ng/ul) ( V1) = (40ng/ul) (5ul)
=.463ul of RNA to make 40ng/ul

qPCR set-up
-master mix, 20ul each
-For each sample 1 ul of template used with concentration 40ng/ul

Sample	Volume ul	Multiplier	Total
So Fast	10ul	x9.9	99.00
FWD	0.5	x9.9	04.95
REV	0.5	x9.9	04.95
H2O	8	x9.9	79.20

-Total 8 samples 1 positive control and 2 neg controls

Order of well placment

Well #	ROW 1	ROW 2
1	CT1 GILL	POSITIVE CONTROL
2	CT1 MANTLE	NO TEMPLATE
3	CT1 MUSCLE	NO TEMPLATE
4	CT1 GONAD
5	EST1 GILL
6	EST1 MANTLE
7	EST1 MUSCLE
8	EST1 GONAD

---

28 February 2012
Summary:
-Ran DNAse Turbo-Free protocol, rigorous treatment.

Procedure:
-Followed DNAse commercial protocol for Roberts Lab.
-Used .5ml tubes for 8 samples, CT1 - EST1.
RNA quantification (ng/ul)

CT1 GILL	229.80
CT1 MANTLE	88.65
CT1 MUSCLE	213.60
CT1 GONAD	941.76
EST1 GILL	1121.69
EST1 MANTLE	601.64
EST1 MUSCLE	438.82
EST1 GONAD	1107.45

-Diluted each sample so I had 5ug per 50 ul.
ex. 10 / .2298 = 21.75
need to add 21.75ul of RNA and 6.5 ul of H2O for desired concentration.

Dilutions

Sample	RNA(ul)	H2O(ul)
CT1 GILL	21.75	28.24
MANTLE	50	0
MUSCLE	23.41	26.59
GONAD	5.31	44.69
EST1 GILL	4.46	45.54
MANTLE	8.31	41.69
MUSCLE	11.39	38.60
GONAD	4.52	45.49

NEXT STEP:
-Run spec on samples

---

22 February 2012
Summary:
-ran PCR for all 32 samples and 2 positive controls for each set of primers

Procedure:
-2 master mixes were made
First: for 37 samples using primer SRID 1346 and 1345 - product length 186
Second: Only for positive control Vg2 primer Product length 700
Table:
-All wells follow order of Gill, Mantle, Muscle, Gonad unless specified.
-Example: wells 2-5 contain CT1 GILL CT1 MANTLE, CT1 MUSCLE, CT1 GONAD
-ALL Primers are for the vitellogenin gene

Row 1	Well 1	2-5	6-9	10-13	14-17	18	19-20
	Hyperladder II	CT1	EST1	CT2	EST2	Vg1 positive control length 186	no template
ROW 2	WELL 1	2-5	6-9	10-13	14-17	18	19-20
	Hyperladder II	CT3	EST3	CT4	EST4	Vg2 positive control length 700	No template

Next Step:
-Run Gel for ER primers

---

21 February 2012
Summary:
-Completed cDNA conversion
-Sample CT3 & 4 and EST3 & 4

Procedure:
-Was completed to lab protocol
-Stored in -20C box

Next Step:
-Run PCR 22 Feb

---

17 February 2012
Summary:
-Completed RNA to cDNA conversion for last 16 samples from experiment
-Samples CT3 & 4 and EST 3 & 4
-Already have primers necessary from July 2011, 186bps (Vitellogenin)

Procedure:
Table: Used 1 ug of RNA per sample
-All procedures were done to protocol

Next Step:
-On 22 FEB run PCR with positive control and both vitellogenin primers
-Run gel on new set of samples
-Begin qPCR if samples and primers are good

---

13 February 2012
Summary:
-Wasn't able to finish RNA isolation today, will finish 14 Feb

Next Step:
-Finish conversion to cDNA
-Run gel with new cDNA
-Design Primers under 200bps

---

08 February 2012
Summary:
-Began RNA isolation

Procedure:
-Began isolation procedure to protocol on CT3 - CT4, EST3 - EST4
-total is 16 samples

---

07 February 2012
Summary:
-Ran gel on 16 samples

-Read gel from right to left, right side being well #1
-Gel turned out nicely, a red X below means a positive result for active vitellogenin gene.

Procedure:
GEL layout - 28 slots - .8% gel, .6g agarose

WELL
1 - HYPERLADDER	11 - EST1 GILL X
2 - POS CONTROL X	12 - EST1 MANTLE X
3 - CT1 GILL	13 - EST 1 MUSCLE X
4 - CT1 MANTLE	14 - EST1 GONAD X
5 - CT1 MUSCLE	15 - EST2 GILL X
6 - CT1 GONAD	16 - EST2 MANTLE
7 - CT2 GILL X	17 - EST2 MUSCLE
8 - CT2 MANTLE	18 - EST2 GONAD
9 - CT2 MUSCLE	19 - NO TEMP
10 - CT2 GONAD	20 - NO TEMP

Master Mix - same as 17 January 2012, except with 20 samples

Well Plate Layout

Well 10	Well 11	Well 12
POS CONTROL	EST1 GILL	CT1 GILL
NO TEMP	MANTLE	MANTLE
NO TEMP	MUSCLE	MUSCLE
	GONAD	GONAD
	EST2 GILL	CT2 GILL
	MANTLE	MANTLE
	MUSCLE	MUSCLE
	GONAD	GONAD

Thermocycler:
Thermocycler parameters:
95°C - 10mins
40 cyles of:
95°C - 15s
55C - 15s
72°C - 10s - 2mins

Next Step:
-Convert the rest of my sample to RNA this week and next week to cDNA
-then run PCR

---

06 February 2012
Summary:
-Ran reverse transcription procedures to protocol.

Procedure:
Table - 1ug total RNA for each sample
ex. 1.0ul/0.08895ug/ul = 11.30ul RNA

SAMPLE	ul RNA	ul H2O
CT1 GILL	4.35	13.4
MANTLE	11.3	6.5
MUSCLE	4.68	13.07
GONAD	1.06	16.69
CT2 GILL	0.78	16.96
MANTLE	1.34	16.41
MUSCLE	1.51	16.23
GONAD	0.83	16.92
EST1 GILL	0.89	16.85
MANTLE	1.66	16.08
MUSCLE	2.28	15.47
GONAD	0.90	16.84
EST2 GILL	1.73	16.01
MANTLE	1.71	16.04
MUSCLE	5.38	12.37
GONAD	0.78	16.97

RNA Reverse Transcription Master Mix

5x Buffer	5ul	x16	80ul
10mM dNTPS	1.25ul	x16	20ul
M-MLV RT	0.5ul	x16	8ul
Total			108ul

Next Step:
-Run PCR 7 FEB

---

02 February 2012
Summary:
-Completed RNA quantification procedures

Procedure:
Table - RNA quantification

SAMPLE	ng/ul	260/280	260/230
CT1 GILL	229.80	1.90	1.15
CT1 MANTLE	88.65	1.75	.33
CT1 MUSCLE	213.60	1.85	1.32
CT1 GONAD	941.76	1.92	0.68
CT2 GILL	1272.62	1.99	1.67
CT2 MANTLE	744.69	1.92	1.01
CT2 MUSCLE	658.71	1.95	1.11
CT2 GONAD	1211.46	1.97	1.07
EST1 GILL	1121.69	1.97	1.89
EST1 MANTLE	601.64	1.97	1.44
EST1 MUSCLE	438.82	1.86	1.46
EST1 GONAD	1107.45	1.90	0.62
EST2 GILL	578.10	1.91	1.33
EST2 MANTLE	585.63	1.86	0.45
EST2 MUSCLE	185.91	1.83	1.61
EST2 GONAD	1290.52	1.98	1.31

SAMPLE	ng/ul	260/280	260/230
CT3 GILL	619.59	2.01	2.02
MANTLE	341.72	2.00	1.83
MUSCLE	111.75	1.81	1.81
GONAD	337.17	1.92	0.69
CT4 GILL	451.76	2.02	1.44
MANTLE	586.33	2.05	1.17
MUSCLE	155.96	1.87	1.54
GONAD	457.95	1.91	0.94
EST3 GILL	1100.32	2.08	1.74
MANTLE	596.05	2.06	1.01
MUSCLE	554.94	2.01	1.49
GONAD	857.73	2.07	0.77
EST4 GILL	717.12	2.04	1.05
MANTLE	256.68	1.94	0.57
MUSCLE	683.52	2.01	1.72
GONAD	567.80	1.93	0.45

Next Step:
-Run reverse transcription procedures

---

31 January 2012
Summary:
-Completed RNA isolation procedures to protocol
-Will begin reverse transcription 6 Feb

Next Step:
-Run PCR on 7 Feb with new cDNA samples

---

30 January 2012
Summary:
-Began RNA isolation procedures for 16 samples from my experiment.

Procedure:
-RNA isolation was done to procedure, a copy can be found here.

Next Step:
-Complete RNA and reverse transcription procedures 31 January
-Run PCR on samples 1 FEB

---

25 January 2012
Summary:
-Ran gel with diluted samples x10, x100, x1000
-A photo of the finished gel can be found here

-Smear is still present within the x10 dilution, also my no templates are contaminated. However, the same pattern is observed as with the 3 previous gels.

Well 1	2	3	4	5	6	7	8	9	10	11	12
Hyperladder II	CT1 Gill x10	"" x100	""x1000	EST1 Gill x10	"" x100	""x1000	EST1 Gonad x10	""x100	""x1000	No temp.	No temp.

Procedure:
-Gel was 8% instead of 1.2% agarose

Next Step:
-Start from the beginning, going to create new RNA and cDNA this week.

---

24 January 2012
Summary:
-Ran gel containing experimental samples and positive control. The positive control turned out again confirming the primer, however the experimental samples came out smeared.
-Going to run gel tomorrow with x10, x100, x1000 cDNA samples.
-Photo of finished gel for experimental samples can be found here.

Well 1	2	3	4	5	6	7	8	9	10	11	12
Hyperladder II	CT1 Gill	" " Mantle	" " Muscle	EST1 Gill	" 'Mantle	" " Muscle	" " Gonad	No temp.	No temp.

Procedure:
Diluted cDNA gel -
-Master mix same as 23 January 2012.
-Thermocycler temperatures and times same as 23 January 2012.
- cDNA diluted by taking 1ul and adding 9ul H2O for x10 etc.

Well Plate location:

Well	6	7
1	CT1 Gill x10	EST1 Gonad x1000
2	" " x100	No template
3	" " x1000	No template
4	EST1 Gill x10
5	" " x100
6	" " x1000
7	EST1 Gonad x10
8	" " x100

Next Step:
-Run gel, if the results are no different then remake cDNA.

---

23 January 2012
Summary:
-Ran PCR testing experimental samples with positive control

Procedure:
-Master Mix same as 17 January 2012 expect x11 quantity.
-Primer used was CgVg2, with a positive band at 700bp being used as the positive control for vitellogenin.

Well	9	10
1	Positive Control (Manel's)	No Template
2	CT1 Gill	No Template
3	CT1 Mantle
4	CT1 Muscle
5	EST1 Gill
6	EST1 Mantle
7	EST1 Muscle
8	EST1 Gonad

Next Step:
-Run Gel

---

17 January 2012
Summary:
-Ran gel utilizing Manel's cDNA
-Positive control may have been found for experiment
-For future reference CgVg2 primers came from "Molecular Characterization of a cDNA Encoding Putative Vitellogenin from the Pacific Oyster Crassostrea gigas" by Matsumoto 2003.

Procedure:
-Made identical master mix's for x2 primers.
1. 1414/1415
F primer: CTCAACAGCCCTGGTGGCGG SR ID: 1414
R primer: AGCGGTTCCGACTGCTCCCT SR ID: 1415
Product size: 723

2. CgVg2
Primer 1 (4) - 5' -GCA GAT GGA AGG ATG TCC ATC AG - 3'
Primer 2 (5) - 5' -TTC ACA GTC ATG GAG CCC AGC AT - 3'
Product size: 700

Template	1 ul	x	x
FWD	0.5ul	x3.3	1.65ul
REV	0.5ul	x3.3	1.65ul
x2 RED	12.5ul	x3.3	41.25ul
H2O	10.5ul	x3.3	34.65ul
Total	25.0ul	x3.3	x

Thermocycler parameters:
95°C - 10mins
40 cyles of:
95°C - 15s
55C - 15s
72°C - 10s - 2mins

Gel - 75ml at 1.2% agarose or 0.9g agarose
Photo copy of the gel can be found here.
Gel layout:

Well 1	2	3	4	5	6	7
Hyperladder II	1414/1415 primer	No template	No template	CgVg2 Primer	No template	No Template

Next Step:
-Run my cDNA samples with sample from today

---

11 January 2012
Summary:
-Reran gel, results suggests an abundance of double stranded DNA.
-Correction - Going to run a separate sample of cDNA from Manel's experiment, this should eliminate whether it is my cDNA samples or my technique that is causing the problem. In addition I will run a second sample with cDNA from a CT2 and EST2 (not yet run) and compare to CT1 and EST1 (inconclusive results).

Procedure:
Ran gel to protocol.
-A copy of the gel can be found here.

Next Step:
-Run Manel's cDNA sample with 1414 and 1415 primer.
-Convert remaining samples to cDNA
-Run CT2 and EST2 cDNA

---

09 January 2012
Summary:
-made master mix
-ran samples through thermocycler
Procedure:
Master mix -for x7 samples and x2 no templates

Template	2ul	-	-
Forward Primer SR ID 1414	0.5	x10	5.0
reverse Primer SR ID 1415	0.5	x10	5.0
x2 Apex Red	12.5	x10	125
Pure H2O	9.5	x10x	95
Totals	25ul	10	230ul

Well plate Layout -

Well #	3	4
1	CT1 Gill	No template
2	CT1 Mantle	blank
3	CT1 Muscle	blank
4	EST1 Gill	blank
5	EST1 Mantle	blank
6	EST1 Muscle	blank
7	EST1 Gonad	blank
8	No template	blank

Thermocycler times -
Thermocycler parameters:
95°C - 10mins
40 cyles of:
95°C - 15s
55C - 15s
72°C - 30 sec

Next Step:
-Run gel

---

06 January 2012
Summary:
-Ran gel containing 1414 and 1415 primers for vitellogenin
Procedure:
-Made a 24 well 150ml gel at .8% agarose and 15ul of EtBR
Gel Layout:

Well #

1

2

3

4

5

6

7

8

9

10

11

12

Row 1

ladder (5ul)

CT1 Gill

CT1 Gill - NT

CT1 Gill - NT

CT1 Mantle

CT1 Mantle - NT

CT1 Mantle -NT

CT1 Muscle

CT1 Muscle -NT

CT1 Muscle -NT

EST1 Gill

EST1 Gill - NT

Row 2

ladder (5ul)

EST1 Gill - NT

EST1 Mantle

EST1 Mantle - NT

EST1 Mantle - NT

EST1 Muscle

EST 1 Muscle - NT

EST1 Muscle - NT

EST1 Gonad

EST1 Gonad - NT

EST1 Gonad - NT

*NT = no template
-A copy of the gel can be found here
Conclusion:
-Gel is no good, contamination is present in every well.
Next Step:
-Rerun gel, only x2 no templates per primer.

---

05 January 2012
Summary:
-Obtained primer SRID's 1414 and 1415
-Made master mix and began thermocycler
Procedure:
-Total 7 tissue samples with 2 non-templates each
-Used 2ul of template
WELL LAYOUT:

	Well 5	Well 6	Well 7
1	CT1 GILL	CT1 MUSCLE NO TEMPLATE	EST1 MUSCLE NO TEMPLATE
2	CT1 GILL NO TEMPLATE	EST1 GILL	EST1 GONAD
3	CT1 GILL NO TEMPLATE	EST1 GILL NO TEMPLATE	EST1 GONAD NO TEMPLATE
4	CT1 MANTLE	EST1 GILL NO TEMPLATE	EST1 GONAD NO TEMPLATE
5	CT1 MANTLE NO TEMPLATE	EST1 MANTLE NO TEMPLATE
6	CT1 MANTLE NO TEMPLATE	EST1 MANTLE NO TEMPLATE
7	CT1 MUSCLE	EST1 MUSCLE
8	CT1 MUSCLE NO TEMPLATE	EST1 MUSCLE NO TEMPLATE

Thermocycler parameters:
95°C - 10mins
40 cyles of:
95°C - 15s
55C - 15s
72°C - 30 sec

Next Step:
-Run gel tomorrow

---

03 January 2012
Summary:
-Will run a gel using Manel's primers for Vitellogenin, which indicated a positive product of 723bps.
Procedure:
- Manel's Primer information
F primer: CTCAACAGCCCTGGTGGCGG SR ID: 1414
R primer: AGCGGTTCCGACTGCTCCCT SR ID: 1415
Product size: 723
Thermocycler parameters:
95°C - 10mins
40 cyles of:
95°C - 15s
55C - 15s
72°C - 30 sec

---

30 September 2011
Summary:
-Ran gel containing CgVg2 Primers.
Procedure:
-Gel was ran to protocol.

Well 1	CT1 Gill CgVg2
2	CT1 Gill CgVg2 - No Template
3	CT1 Gill CgVg2 - No Template
4	CT1 Mantle CgVg2
5	CT1 Mantle CgVg2 - No Template
6	CT1 Mantle CgVg2 - No Template
7	EST1 Gill CgVg2
8	EST1 Gill CgVg2 - No Template
9	EST1 Gill CgVg2 - No Template
10	EST1 Mantle CgVg2
11	EST1 Mantle CgVg2 - No Template
12	EST1 Mantle CgVg2 - No Template
13	EST1 Gonad/ D.G. CgVg2
14	EST1 Gonad/ D.G. CgVg2 - No Template
15	EST1 Gonad/ D.G. CgVg2 - No Template
16	Hyper Ladder 1

-Photo of finished gel can be found here.

---

16 September 2011
Summary:
-Ran gel containing CgER2 primer.
Procedure:
-Gel was ran to protocol.

Well 1	Hyperladder 1
2	CT1 Gill ER2
3	CT1 Gill CgER2 - No Template
4	CT1 Gill CgER2 - No Template
5	CT1 Mantle ER2
6	CT1 Mantle CgER2 - No Template
7	CT1 mantle CgER2 - No Template
8	EST1 Gill ER2
9	EST1 Gill CgER2 - No Template
10	EST1 Gill CgER2 - No Template
11	EST1 Mantle ER2
12	EST1 Mantle CgER2 - No Template
13	EST1 Mantle CgER2 - No Template
14	EST1 Gonad/ D.G. ER2
15	EST1 Gonad/D.G. CgER2 - No Template
16	EST1 Gonad/D.G. CgER2 - No Template

-Photo of finished gel can be found here.

Next Step:
-Run CgVg2 Gel.
-Write Proposal.

---

14 September 2011
Summary:
-Ran PCR for 13 Sep 2011 samples A1-F1.
Procedure:
Table for Sample Placement

Well #	Sample	Tissue	Primer Type	Temp	Controls
3	CT1	Gill	CgER2	A	B & C
4	CT1	Mantle	CgER2	A	B & C
5	EST1	Gill	CgER2	A	B & C
6	EST1	Mantle	CgER2	A	B & C
7	EST1	Gonad/ D.G.	CgER2	A	B & C
8	CT1	Gill	CgVg2	A	B & C
9	CT1	Mantle	CgVg2	A	B & C
10	EST1	Gill	CgVg2	A	B & C
11	EST1	Mantle	CgVg2	A	B & C
12	EST1	Gonad/ D.G.	CgVg2	A	B & C

Thermo-cycler times:

1. 95C	10 mins
2. 95C	15 secs
3. 60C	15 secs
4. 72C	15 secs
5. 72C	10 mins

Next Step:
-Run gel

---

13 September 2011
Summary:
-Ran reverse transcription procedure on 30 August tissues.
-Re ran CT1 Muscle; CT1 Gonda/Digestive Gland; and EST1 Muscle tissues through RNA isolation procedures.
Procedure:
-All procedures were ran to protocol.

Location	Name	RNA (ul)	H2O (ul)
A1	CT1 Gill	2.20	15.60
B1	CT1 Mantle	4.60	13.20
D1	EST1 Gill	4.0	13.70
E1	EST 1 Mantle	2.4	15.30
F1	EST 1 Gonad/D.G.	6.0	11.75

-EST1 Gonad/D.G. was diluted with nano H2O then added to respective well.
RT Master Mix:

Name	(ul)	RXN	+10%	Final Total (ul)
x5 Buffer	5	5	.1	27.5
dNTPs	1.25	5	.1	6.88
RT Primer	.5	5	.1	2.75

Next Step:
-Run gel to determine if I am getting a response.
-Finish proposal for senior capstone.

---

12 September 2011
Summary:
-Finished calculations for reverse transcription of first five tissues.
-Began proposal for senior capstone.
Next Step:
-Run RT procedure.
-Run Gel
-Finish proposal.

---

30 August 2011
Summary:
-Completed RNA extraction for samples CT1 and EST1.
-CT1 samples for a single specimen completed. Total 2 samples (gill, mantle)
-EST 1 samples for a single specimen were completed. Total 3 samples (gill, mantle, gonad/digestive gland)
-Couldn't separate supernatant from CT1 (muscle, gonad/digestive gland) and EST1 (Muscle). These three were place in the -80C box for storage.
-It was determined after the last PCR ran that ER2 and Vg2 will be used for identification of the genes.
Procedure:
-RNA extraction was ran to protocol.
-Added 50ul to each tube except gonad added 75ul.

Tissue	ng/ul
CT1 Gill	463.4
CT1 Mantle	218.0
EST 1 Gill	248.0
EST 1 Mantle	410.5
EST 1Gonad/D.G.	1685.7

Next Step:
-Convert RNA to cDNA
-Run PCR on all samples from specimens CT1 and EST.

---

17 August 2011
Summary:
-Completed PCR for ER1, ER2, Vg1, and Vg2 primers.
-Copy of gel can be found here.

Well 1	2	3	4	5	6	7	8	9	10	11	12
Ladder 1 (10k-200)	ER1 Control	ER1 Primer ~200bp	Blank	ER2 Control	ER2 Primer ~200bp	Blank	Vg1 Control	Vg1 Primer ~200bp	Blank	Vg2 Control	Vg2 Primer ~900bp

-At 1205 began dissection of oyster tissues. Controls were dissected first.
Procedure:
-Tissue collection: Total of eight oysters at 4-5 tissues selected from each oyster.
1. Gill 2. Mantle 3. Muscle 4. Gonad/(5.) Digestive gland
-The gonad and digestive gland were combined.
-All procedures were done to protocol.
-Tubes were labeled with date/ Control (green) or Estradiol (orange)/ Oyster Number/ tissue type. Stored in -80C freezer.
Next Step:
-Verify PCR
-Start extraction

---

16 August 2011
Summary:
-Experiment began at 1205
Procedure:
-Setup:
x2 tanks (5L) each were filled with 2L seawater.
Airstones and pump supply O2.
Lids were placed over both tanks.
Oysters were weighed dry before being placed in respective tanks x4 C.gigas.
Total for Control Tank (384.79g) Total for Tank (E) (459.88g)
Each tank was given ~2mL of Instant Algae
Control tank: added 100uL Ethanol, Tank (E): Added 100ul Solution for a concentration of 50ng/L 17 Alpha-Ethinylestradiol.
Exposure to run 24hours.
Next Step:
-Run PCR
-Collect tissues in 24hours.

---

15 August 2011
Summary:
-Need to retest all primers again with higher annealing temperature (60C)
-Lengths for primers cgER2 (>200bp) and cgVg2 (~900bp).
-Designed experiment for quick trial
Tank 1 (2L): Control, x4 C.gigas with 100ul Ethanol added.
Tank 2 (2L): Tank (E), x4 C.gigas with 100ul solution. Total concentration is at 50ng/L of 17 Alpha-Ethinylestradiol, the estradiol was dissolved in 100% ethanol.
-Obtained x8 oyster (C.gigas)
Procedure:
-To calculate concentration needed I referenced Andrew, 2008 (50ng/L)
-100mg of 17 Alpha-Ethinylestradiol added to 10mL Ethanol. Equation (100mg/.01L) (V1) = (5e^-5) (2L), V1 = 100uL solution.
-Planned exposure is 24hours.
Next Step:
-Set up Experiment
-Need to retest all primers again with higher annealing temperature (60C)

---

10 August 2011
Summary:
-made master mixes for new primers cgER2 and cgVg2.
-Ran PCR.
-Ran gel a finished photo can be found here. Close up.

Hyper ladder 1	ER Control	cgER2	Vg Control	cgVg2
>200

Procedure:
-Master Mix for cgER2

Template	2ul	n/a	n/a
Primer FWD SRI key:	0.5ul	x2.2	1.1ul
Primer REV SRI key:	0.5ul	x2.2	1.1ul
x2 Apex Red	12.5ul	x2.2	27.5ul
Nano H2O	9.5ul	x2.2	20.9ul
Totals	25ul	n/a	50.6ul

-Master Mix for cgVg2

Template	5ul	n/a	n/a
Primer FWD SRI key:	0.5ul	x2.2	1.1ul
Primer REV SRI key:	0.5ul	x2.2	1.1ul
x2 Apex Red	12.5ul	x2.2	27.5ul
Nano H2O	6.5ul	x2.2	14.3ul
Totals	25ul	n/a	44.0ul

-PCR times: 1 cycle 95C 10mins, 39 Cycles 95C 15s, 55C 15s, 72C 30s, 1 cycle 72C 10mins
-Gel was made of 1.2% agarose. 75ml gel with .9g agarose.
Next Step:
-Design experimental setup

---

9 August 2011
Summary:
-Ordered new primers for ER and Vg as outlined in Matsumoto. T et al, 2003/2007.
-CgER2 - FWD 5'-CCT ACT CGA CCC CTC CCT ATC - 3' 21bp
-CgER2 - REV 5'-CAC CCC TCA CAT GAC CAC AC - 3' 20bp
-CgVg2 - FWD 5'-TTC ACA GTC ATG GAG CCC AGC AT -3' 23bp
-CgVg2- REV 5'-GCA GAT GGA AGG ATG TCC ATC AG -3' 23bp
-Ordered 17 Alpha-Ethinylestradiol 100mg.
Next Step:
-Run new primers
-Design experimental setup

---

2 August 2011
Summary:
-Vitellogenin is produced within the ovary's of female C. gigas (Matsumoto T., 2003) Also from this paper 2 primers were identified and will be ordered.
Primer 1 (4) - 5' -GCA GAT GGA AGG ATG TCC ATC AG - 3'
Primer 2 (5) - 5' -TTC ACA GTC ATG GAG CCC AGC AT - 3'
-The thermocycler times used were also different within the Matsumoto, 2003 journal and consisted of 1 cycle 92C 1min, 40 cycles 92C 1min, 55C 1min, 72C 1min, and 1 cycle 72C 5min. These temperatures may be used for next PCR.
-Circulation of estrodial-17B flows significantly toward the digestive glands of C. gigas where a large percent is converted to estrone by 17B-HSD. (Le-Curiurx Belfond O., 2005).
-Ran Gel with various tissues from C. gigas ( mantle, gill, muscle, gonads, digestive gland (none sex specific). Photo of Gel can be found here.
-Results varied from test with only mantle tissue, the ER primer presented a band <200bp and another around 700bp. The ER control was negative for product. The VTG primer shows many bands ranging from 700bp to < 200bp and the control also shows contamination.
Procedure:
-Ran gel to protocol.
Next Step:
-Order primers for VTG (Vg)
-Run primers with 5ul of template
-Research work done on VTG expression.
-Design Experiment
-Collect oysters

---

1 August 2011
Summary:
-Made Separate master mixes for ER and VTG primers.
-made gel with 0.9g agarose
Procedure:
-Same master mix procedure used as 13 July 2011, only change was template used.
-Samples ran at 95C-15s, 55C-15s, 72C-30s.
Next Step:
-Run electrophoresis

---

22 July 2011
Summary:
-ER and VTG controls were both negative. Photo found on 13 July 2011 link.
-The ER primer gave a positive product band below 200bp.
-The VTG primer, a slight band occurred, small product most likely due to the tissue used. The mantle tissue used originally would not likely have high expression since VTG is an egg-yolk precursor.
Next Step:
-Run VTG primers with multiple tissues.

---

13 July 2011
Summary:
-Made separate master mixes for ER and VTG primers.
-Ran Electrophoresis on samples.
-Copy of completed gel can be found here.
Procedure:
-Master mixes x2 were made as follows.

Template	2ul	n/a	n/a
x2 Apex Red	12.5ul	x2.2	27.5ul
Primer FWD ER SRI Key -1343 VTG SRI Key - 1345	0.5ul	x2.2	1.1ul
Primer REV ER SRI Key -1344 VTG SRI Key - 1346	0.5ul	x2.2	1.1ul
Nano H2O	9.5ul	x2.2	20.9ul
Total	25ul	n/a	50.6ul each MM

-Well Plate Configuration.
-Added 23ul of appropriate master mix to each well.
-Then added template (2ul) (cDNA from C1 Crassostrea gigas) and control H2O (2ul).

Well Number	10
Neg ER Control	added 2ul nano H2O
ER Sample	added 2ul of C1
Neg VTG Control	added 2ul nano H2O
VTG Sample	added 2ul of C1

-Thermal Cycle was done to protocol except 72C was extended to 30secs per cycle.
Next Step:

---

12 July 2011
Summary:
-Researched past studies involving contraceptive chemicals and aquatic species.
-Researched active ingredient of birth control, there are many variations of the active ingredient between BC's.
-A common synthetic estrogen used in studies is EE2 (17a-Ethinylestradiol), Kidd et al, 2010., Wessel et al, 2007.
Next Step:
-Run ER & VTG primers

---

11 July 2011
Summary:
-Researched mammalian estrogen pathways
-Ran BLAST for Crassostrea gigas Estrogen Receptor cds, Accession # HS180695.1
-Ran BLAST for C. gigas Vitellogenin cds, Accession # AB084783
-Designed Primers for ER (SRI Keys 1343 & 1344) and Vitellogenin (SRI Keys 1345 & 1346)
Procedure:
-Coding sequences taken from NCBI/Genbank
-Primers designed with Geneious
Next Step:
-Run primers to test for product
-Research main compounds of synthetic birth control

---

01 July 2011
Summary:
-Ran electrophoresis gel
Procedure:
-Photo of finished gel
Next Step:
-Research experimental design

---

24 June 2011
Summary:
-Made agarose gel
Procedure:
1. Used 75ml of TAE
2. At .8% agarose, .6g agarose for 75ml solution
3. Weighed (278g) then heated for 2 min till all particulate disappeared
4. Removed swirled and added nano water to replace evaporated solution
5. Added 7.5uL Ethidum Bromide to solution
6. Waited to room temperature, poured gel
7. Wait 20min till hardened
Next Step:
-Run gel

---

20 June 2011
0900-1500
Summary:
-Began PCR procedure to protocol.
-Researched estrogenic effects on C. gigas for proposal
-Reviewed transcription processes
Procedure:
-Made master mix as following

Template	2ul	n/a	n/a
2x APEX	12.5ul	x6	75ul
Defensin Primer SRI-KEY 1070 & 1109	0.5ul	x6	3ul
Nano H2O	10ul	x6	60ul
Totals for Master Mix	25ul		138ul

-Placed into well plate with 23ul each of master mix, as following

Row	Column 3
1	Sample C1
2	Sample C2
3	RT Control
4	PCR Control
5	PCR Control

-Cycling parameters used:

95C	10min
40 cycles of
95C	30sec
50C	30sec
72C	2min 30sec

-Placed container into -20C box.
Next Step:
-Create and run electrophoresis

---

27 May 2011
Summary:
-Ran reverse transcription procedures to protocol for samples C1, C2, and control.
-Placed samples in -20C freezer.

Next Step:
-Perform PCR procedures

---

24 May 2011
Summary:
-Finished mrna isolation procedure to protocol.
-ran spec on samples.
-Made -80C box and placed samples on top shelf.
Procedure:
-spec was run using Nanodrop

Sample	ng/ul	A260	A280	260/280	260/230	Constant	Cursor Pos.	Cursor Abs.	340 raw
C1	374.10	9.352	4.852	1.93	1.60	40.00	230	5.828	0.379
C2	956.07	23.902	12.254	1.95	1.16	40.00	230	20.595	1.766

Next Step:
-Start cdna procedures

---

23 May 2011
Summary:
-Began mrna isolation procedures using Crassostrea gigas tissue.
Procedure:
-mrna isolation was done to protocol.
Next Step:
-Next step would be to treat with DNAase.
-make cdna from rna and then perform pcr on samples.

---

12 May 2011
Summary:
-Ran samples from 04 May 2011
Procedure:
-Loaded gel with marker of known size close to that of defensin gene.
-Loaded gel with control, DH, and BB samples.
-Ran electrophoresis for 25min.
-Examined under UV light
-Here is a foto of the finished gel
Next Step:
-Repeat procdure for practice.
-Move onto rna isolation procedures.

---

09 May 2011
Summary:
-Poured electrophoresis gels
Procedure:
-Decide volume of gel needed, add to flask.
-Add 0.08% agarose, weigh flask, heat sample to boiling, remove and swirl to remove any particulates, reheat if necessary.
-Place on scale replace evaporated mix with nano water, add about 1 gram more then previous weight to account for evaporation.
-Place solution into gel mold, wait 30min.
Next Step:
-Ran out of time, placed completed gel into the fridge. Tomorrow finish electrophoresis.

---

04 May 2011
Summary:
-Copy of master mix can be found here.
-Started PCR working with gigas cDNA.
Procedure:
-Defensin primers SRI-Key (1070 and 1109)
-Started by producing a master mix to fill a total of 3 wells (2 cDNA, 1 control) with 23uL each, I added 10% to the mix to account for errors, see link above for exact concentrations.
-Well A- control and was filled with 2uL of PCR water, Well B- Dreyton Harbor sample added 2uL, Well C- Big Beef Creek sample added 2uL.
-Placed well plate into thermal cycler.
Next Step:
-Learn to pour agar gel plates and perform electrophoresis on samples.

---

27 April 2011
Summary:
-Reviewed Rna isolation procedures.
-Completed BioSafety Training and emailed copy of cert to Sam.

Next step:
- Next week work on rna isolation.