20130510

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Designed the following C.gigas primers for MeDIP PCR. All primers had at least 1 CG dinucleotide in them.

Primer name	Gene	Gene Accession#
Cg_MAPK_MeDIP_F	Mitogen-activated protein kinase 7	CGI_10020574
Cg_MAPK_MeDIP_R	Mitogen-activated protein kinase 7	CGI_10020574
Cg_CATHB_MeDIP_F	Cathepsin B	CGI_10027022
Cg_CATHB_MeDIP_R	Cathepsin B	CGI_10027022
Cg_CATA_MeDIP_F	Catalase	CGI_10003355
Cg_CATA_MeDIP_R	Catalase	CGI_10003355
Cg_CAP_MeDIP_F	Caspase - 3	CGI_10015439
Cg_CAP_MeDIP_R	Caspase - 3	CGI_10015439
Cg_SOD_MeDIP_F	Superoxide dismutase [Cu-Zn], chloroplastic	CGI_10025077
Cg_SOD_MeDIP_R	Superoxide dismutase [Cu-Zn], chloroplastic	CGI_10025077

20130503

Ran qPCR on the following samples of C. gigas using primers Cg_HSP70_cm_f1 (SRID: 971) and Cg_HSP70_cm_r1 (SRID 972).

Single reaction (20uL) set up is listed below.

Mastermix was made of 10 uL 2X Sso Fast EvaGreen, 0.5 uL of 10 µM Forward Primer, 0.5 uL of 10 µM Reverse Primer, 8 uL Nano Pure Water.


Lane 1 = NTC
Lane 2 = NTC
Lane 3 = NTC
Lane 4 = Exp 1 - 2, 1 uL
Lane 5 = Exp 1 - 12, 1 uL
Lane 6 = Exp 1 - 26, 1 uL
Lane 7 = Exp 1 - 138, 1 uL
Lane 8 = Exp 1 - 146, 1 uL
Lane 9 = Exp 1 - 158, 1 uL

Centrifuged qPCR plate for 1 min @ 3000g.

Put in thermalcycler. Cycling paramaters:

1) 95C - 2 min
2) 98C - 2 sec
3) 55C - 5 sec
4) Plate Read
5) Go to line 2, 39 times
6) 75C - 10 sec
7) Melt Curve from 55C to 95C, read every 0.2C, hold 10 sec

Results: All samples amplified.

20130502

Discovered three different accession numbers for HSP70 in the Robert's Lab Database. Blasted all three separately. Each accession number matches almost perfectly with at least one other entry for HSP70/HSP in GenBank. Sam said that the extremely high homology amongst HSP70's in GenBank makes its use a bad idea. BLAST results are shown below:

https://www.evernote.com/shard/s256/sh/52fc77c3-74ae-426c-a966-1238329eca7c/0a9bfb1f4fcbbafd6c93b35593cc91ce

20130501

Prepared Reagents for MeDIP:

5x Buffer, 10 mL Solution
50 mM Na2HPO4 (0.5 M Na2HPO4* X = 50 mM Na2HPO4 * 10 mL, X = 1 mL = 1000 uL, 0.5 M Na2HPO4)
700 mM NaCl (5 M NaCl * X = 700 mM NaCl * 10 mL, X = 1.4 mL = 1400 uL, 5 M NaCl)
0.25% Triton-x 100 (0.0025 * 10 mL = 0.025 mL = 25 uL, 0.25% Triton-x 100) [pipette slowly]

1x Buffer, 10 mL Solution
(5x Buffer * X = 1X Buffer * 10 mL, X = 2 mL) Use 2 mL of 5x Buffer + 8 mL H2O)


Digestion Buffer, 5 mL Solution
50 mM Tris HCl, pH = 8.0 (100 mM Tris HCl * X = 50 mM Tris HCl * 5 mL, X = 2.5 mL = 2500 uL 100 mM Tris HCl)
10 mM EDTA, pH = 8.0 (0.5 M EDTA * X = 10 mM EDTA * 5 mL, X = 100 uL, 0.5 M EDTA
0.5% SDS (Prepared 50 mL of a 20% SDS Solution by combining 10 g SDS + 50 mL H2O; add 0.025 mL = 25 uL, 20% SDS)

20130425

Results of PCR Performed on 20130424
Ran 20 uL of each C. gigas sample through agarose gel electrophoresis

TAE 100 mL
Agarose 0.93 g
EtBr 10 uL
Hyper Ladder II, 5 uL
Ran gel electrophoresis for 35 minutes at 120 volts.

Results:

All samples show high intensity bands ~95 bp.

https://www.evernote.com/shard/s256/sh/7c8097d1-be5b-42ba-bf33-03bf2c1331f8/15a3034dce69d4e1c7f5c2198fcda38a



Gel Layout:

Lane 1 - Hyper Ladder II
Lane 2 - Negative Control
Lane 3 - Negative Control
Lane 4 - Negative Control
Lane 5 - Exp 1 - 2
Lane 6 - Exp 1 - 12
Lane 7 - Exp 1 - 26
Lane 8 - Exp 1 - 138
Lane 9 - Exp 1 - 146
Lane 10 - Exp 1 - 158

The following results were given after running the primers Cg_HSP70_cm_f1 (SRID: 971) and Cg_HSP70_cm_r1 (SRID 972) through NCBI Blast:
*Will post on google + to inquire about these blast results.

https://www.evernote.com/shard/s256/sh/ca72411b-db7c-46a9-841f-f22a5da7eec9/e80d21b310852255d57f2d2b359e6a5e

20130424

PCR - C. gigas

Performed Conventional PCR to amplify the following samples of C. gigas using primers Cg_HSP70_cm_f1 (SRID: 971) and Cg_HSP70_cm_r1 (SRID 972).
Exp 1 - 2, 2 uL
Exp 1 - 12, 2 uL
Exp 1 - 26, 2 uL
Exp 1 - 138, 2 uL
Exp 1 - 146, 2 uL
Exp 1 - 158, 2 uL
Mastermix was made of 12.5 uL 2X Apex Red, 0.5 uL of 10 µM Forward Primer, 0.5 uL of 10 µM Reverse Primer, 9.5 uL Nano Pure Water with 2 µL DNA from C. gigas samples.
Thermocycler profile was 95°C 10 min; 40 cycles of 95° for 30 sec, 55° for 30 sec, 72° 30 sec; 72° 10 min.
Held overnight at 4°

Prepared Reagents for -MEDIP

MEDIP 5X Buffer was made of 0.8144 g Sodium Phosphate, 4.0766 g NACL, 25 mL Triton X, filled to 10 mL with water.
MEDIP 1X Buffer was made of 4 mL water, 1 mL MEDIP 5X Buffer.
Digestion Buffer was made of 5 mL water, 0.4006 g Tris HCL brought to 8.02 pH, 0.1436 g EDTA brought to 8.01 pH, and 0.0028 g SDS.

20130419

Examined the Quality of Extracted DNA from C. gigas, along with 6 other Sample From Emma

Ran the following samples through agarose gel electrophoresis with 4 ul of 5X Loading Dye :
Exp 1 - 2, 5 uL
Exp 1 - 12, 10 uL
Exp 1 - 26, 15 uL
Exp 1 - 138, 5 uL
Exp 1 - 146, 5 uL
Exp 1 - 158, 5 uL
C. gigas - 101B2, 2 uL
C. gigas- 101B5, 2 uL
C. gigas- 101B8, 2 uL
C. gigas- 103B224, 2 uL
C. gigas- 103B227, 2 uL
C. gigas- 103B230, 2 uL

TAE 200 mL
Agarose 1.6 g
EtBr 20 uL
Hyper Ladder II 5 uL
Ran gel electrophoresis for 35 minutes at 120 volts.

Results:

All samples displayed high molecular weight bands with no smearing.

https://www.evernote.com/shard/s256/sh/594e28e3-50ae-4fc7-9e1d-618ba1f8c089/8015eb899d1ec4171cce6928f60aaf2d

20130418

DNA Extraction - C. gigas (Gill?) Tissue Samples (Obtained From Emma)

Extracted DNA from the following Pacific Oyster samples using DNAzol:
Exp 1 - 2
Exp 1 - 12
Exp 1 - 26
Exp 1 - 138
Exp 1 - 146
Exp 1 - 158

1. Homogenized ~40 mg tissue in 1 mL DNAzol with a sterile pestle in a 1.5 mL sterile microfuge tube.
2. Incubated samples at room temperature (RT) for 5 minutes.
3. Centrifuged samples for 10 minutes at 10,000 g.
4. Added 0.5 ml of 100% ethanol, then inverted tubes 8 times to form a homogeneous solution.
5. Stored samples at RT for 60 minutes.
6. Centrifuged samples for 5 minutes at 5,000 g.
7. Removed supernatant with pipette, then washed DNA precipitate with 1.0 ml of 75% ethanol.
8. Inverted tubes 5 times to form a homogeneous solution.
9. Centrifuged samples for 2 minutes at 1,000 g.
10. Repeated steps 7-9.
11. Removed ~90% of the alcohol using a pipette, then centrifuged the samples for 2 minutes at 1,000 g.
12. Removed remaining alcohol using a pipette and added 200 mL of Nano Pure H2O to each sample.
13. Stored samples at RT for 25 minutes.

Quantified DNA using Nanodrop spectrophotometer, which gave the following results:

https://www.evernote.com/shard/s256/sh/caaaecba-c01f-4a13-b629-bd85d7916203/388d4173d163eac26060c132a347bb9c