DNA Isolation - Mackenzie’s C.gigas EE2 Gonad Samples

Isolated DNA from the following samples, provided by Mackenzie:

• EE2v2, 22.go

• EE2v2, 20.go

• EE2v2, 28.go

• EE2v2, 29.go

• EE2v2, 16.go

• EE2v2, 32.go

• EE2v2, 24.go

• EE2v2, 33.go

Samples were suspended in 500uL of DNazol (Molecular Research Center), 5uL of PolyAcryl Carrier (Molecular Research Center), 2.75uL Proteinase K (Fermentas; 18.5mg/mL stock), briefly vortexed and incubated 24hrs at RT on rotator. Samples were briefly vortexed and insoluble material was pelleted 10,000g, 10mins, RT. Supe was transferred to fresh tube, mixed with 250uL of 100% EtOH, incubated at RT 5mins, and DNA was pelleted by spinning samples 5,000g, 5mins, RT. Supe was discarded, pellets washed with 1mL of 70% DNazol/30% EtOH solution. Supe was discarded and pellets were washed with 1mL 70% EtOH. Pellets were stored @ -20C under 95% EtOH over the weekend. Supe was discarded and pellets were washed with 70% EtOH. This step was repeated 2 more times. Supe was discarded and pellets were resuspended in Low TE Buffer, spec’d on NanoDrop1000 and run on a gel (10uL of each sample).

Results:

Yields look good and OD260/280 values look excellent. Most of the OD260/230 values aren’t good, but they rarely are.

Gel Loading:

Lane 1 - Hyperladder I (Bioline)

Lane 2 - EV2 16.go

Lane 3 - EV2 20.go

Lane 4 - EV2 22.go

Lane 5 - EV2 24.go

Lane 6 - EV2 28.go

Lane 7 - EV2 29.go

Lane 8 - EV2 32.go

Lane 9 - EV2 33.go

Lane10- Hyperladder I (Bioline)

All samples (excluding EV2 22.go) look pretty good, with minimal smearing. All samples exhibit low molecular weight smear which is either degraded DNA or residual RNA carryover. EV2 22.go had very little tissue, so yields were expected to be extremely low. However, I was anticipating to be able to visualize it on the gel (loaded 10uL = ~90ug).