DNA Extraction Attempt 2 5-12-14

Attempting to extract DNA from 5-10 mm whole oysters minus the shell. This is needed so that sequencing can occur in the future with completed extractions. Unlike the last attempt I will be switching out 95% EtOH for Isopropanol to get better extraction results.

Protocol is as follows:

Incubation for 24 hours at room temp:

1	ml	DNAzol
2.35	ul	Proteinase K (100ug/ml)
1	whole	oyster 5-10 mm
X5		Reactions

1. Pipette 1 ul DNAzol and 2.35 ul Proteinase K in tube containing oyster
2. Homogenize each oyster with disposable pestle.
3. Centrifuge briefly
4. Incubate overnight at ROOM TEMP.

Isolation:

500	ul	Isopropanol
1	ml	75% EtOH
1	ml	75% EtOH
200	ul	Sterile H2O

1. Centrifuge @ 10,000 g for 10 minutes.
2. Collect and Transfer Supernant; Discard pellet
3. Add 500 ul Isopropanol
4. Mix through inversion
5. Incubate for 1 minute at ROOM TEMP.
6. Spin down DNA precipitate
7. Remove supernant
8. Wash with 1 ml 75% EtOH
9. Spin down
10. Remove supernant
11. Wash with 1 ml 75% EtOH
12. Spin down until solid pellet
13. Elute with 200 ul Sterile H2O
14. Quant.