Done to verify removal of gDNA from RNA. Used H.crach_16s_syb_f/r primers. PCR workup/plate layout is here.
Results: Still f’ing gDNA! I’m pretty convinced that this is indeed due to the Ambion kit I’m using being old. Got mixed up with a newer kit, but neither had dates. Mac is going to be running a qPCR later today on DNased RNA that used the other Ambion kit. I will wait until the results of her qPCR to proceed.
Had to re-treat these samples due to residual gDNA that was NOT eliminated the first time through. Possible old Ambion kit??
Inoculated 1x 1L of MB + biphenyl crystals from frozen stock. Incubated 28C, 200RPM. MB was autoclaved with the biphenyl crystals.
Done to verify removal of gDNA from RNA. Used H.crach_16s_syb_f/r primers. PCR workup/plate layout is here.
Results: RNA is still contaminated. Will have to re-DNase the samples.
RNA from yesterday was treated according to Ambion Turbo DNA-free protocol to remove gDNA contamination. Work up is here.
RNA was isolated from the following samples using the MoBio RNA PowerSoil Kit according to protocol:
Results: RNA looks fabulous (as always from the PowerSoil kit). Will DNase treat the samples tomorrow.
RNA from yesterday was treated according to Ambion Turbo DNA-free protocol to remove gDNA contamination. Work up is here. Will check for residual gDNA contamination once I’ve finished with another set of RNA isolations from the remainder of Abalone Dg tissue.
This is a repeat of yesterday’s PCR with a fresh working stock in hopes of eliminating the source of contamination seen in the negative control yesterday.
Lane 1 – 100bp ladder
Lane 2 – 5/13 DNA
Lane 3 – 5/26 DNA
Lane 4 – Empty
Lane 5 – H2O
Results: Same problem as yesterday. Water sample is contaminated. This suggests that the primer stocks are contaminated. Likely due to the use of non-sterile, “old” TE for reconstitution of the primer stocks. Will re-order them and reconstitute them sterily.
Isolated RNA using the MoBio RNA PowerSoil Kit according to protocol from the following samples:
06:6-45
06:6-49
06:6-50
06:6-51
06:6-52
08:4-6
08:4-9
08:4-10
Results: RNA looks fabulous (as always from the PowerSoil kit). Will DNase treat the samples tomorrow.
Performed PCR as set up here using universal bacterial 16s primers (sequences provided by Sara Kelly).
Lane 1 – 100bp ladder
Lane 2- 5/13 DNA
Lane 3 – 5/26 DNA
Lane 4 – H2O
Results: Contamination in the water sample. Hopefully this is just bad technique (never thought I’d say that about myself) and not general, bacterial contamination in the primer stocks, since the stocks (nor the PCR rxns) were prepared steriley. Will repeat.
