Restriction digests from yesterday were mixed with equal volume (50uL) of phenol:chloroform:IAA (25:24:1) and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to a clean tube and an equal volume (50uL) of chloroform was added. Samples were mixed and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to clean tubes and EtOH precipitated, according to protocol. Samples were resuspended in 25uL of H2O and spec’d.
Samples are labeled as Parent (P), #, tissue, enzyme (MspI = M, HpaII = H, Undigested = U)
Results:
Here is a link to a spreadsheet with ODs. Average recovery was ~734ng, which is only a 36% recovery (started with 2000ng). Will need to discuss with Mac and Steven to see if it’s worth continuing with these sample through a second round of digests/phenol:chloroform extraction/EtOH precipitation, as I don’t know what quantity of DNA is required/desired for the subsequent methylation specific PCR (MSP), OR if I should/need to perform a repeat of these 1st-round digestions in order to end up with sufficient DNA for MSP.
