Samples were pelleted at 100g, 4C for 10mins. The supe was mostly removed, leaving ~50uL of supe above the pellets. RNA was isolated according to the TriReagent protocol from the following samples:
3226:
RNAs were resuspended in 10uL of 0.1% DEPC-H2O. They will be DNase treated.
This is a repeat of the previous qPCR from earlier today, BUT I think I might have used the wrong primers in the earlier qPCR (see below). Set up qPCR with the correct (I’m 100% sure of this) primers. Plate layout/workup is here.
Results: Well, in retrospect it looks like I DID use the correct primers earlier! However, the problem is the same. But, the melting curves in the H2O-only samples don’t seem to be the same as what is being seen in the RNA samples, suggesting that the signal in the H2O-only samples are likely primer dimers (melting curve peaks are shifted to the left and are VERY low signals; barely above background).
So, what to do now? Mac has a mad ea suggestion to spike some water with gDNA and DNase treat the sample to assess whether or not the Dase treatment is actually working or not. I think I’ll do this.
Set up qPCR. Plate layout/workup is here.
Results: Looks like gDNA contamination is still present!! This is insane! However, the two water-only samples produced a signal suggesting that something else is contaminated. Will try just qPCR-ing water to see if I can get a clean signal. Will use “store-bought” PCR water instead of NanoPure water.
*UPDATE**: Possibly used 16s universal bacterial primers instead of H.crach 16s primers! Doh! Will re-qPCR using the correct primers.
This is the 3rd time this will be performed on these samples. Hopefully using a different (i.e. new) kit will work. Not sure what’s going on here. Performed according to protocol.
This is a repeat of yesterday’s PCR due to the presence of bands in the water-only samples. Will use reagents and universal 16s bacterial primers (27F & 1492R) provide by the Horner-Devine lab in hopes of: 1) getting this two work and, 2) figuring out the source of the contamination.
All rxns were prepared sterily and all instruments, racks, tubes, tips and water were UV-sterilized for ~45mins in the biological hood. Rxns were prepared in the biological hood. PCR setups are here. Anneal 60C. Cycling params same as yesterday.
Lane 1 – 100bp ladder
Lane 2 – DNA (HD Rxn 1)
Lane 2 – H2O (HD Rxn 1)
Lane 3 – H2O (HD Rxn 1)
Lane 4 – DNA (HD Rxn 2)
Lane 5 – H2O (HD Rxn 2)
Lane 6 – H2O (HD Rxn 2)
Lane 7 – DNA (SR Rxn)
Lane 8 – H2O (SR Rxn)
Lane 9 – H2O (SR Rxn)
Lane 10 – 100bp ladder
Results: Well, we got our band and NO contamination in any H2O lanes. The super-bright, 1500bp band will be excised and purified using Millipore spin columns and submitted for sequencing. However, this gel is interesting because the primers provided by Mike (used in HD Rxn 1 and SR Rxn) did not amplify anything…
Previous PCRs from 20090601 & 20090602 both showed contamination in the negative control. Suspect that the primer stocks were contaminated due to the usage of older, non-sterile TE for reconstitution. New stocks were received and reconstituted with filter-sterilized TE. Working stocks were made with filter-sterilized Nanopure H2O. All pipettes, tips, tubes, racks were UV-sterilized in the biological hood. The PCR reaction was set up in the biological hood. PCR set up is here. Used universal 16s bacteria primers (27F, 1492R). Sequences from Sara Kelly. Anneal 60C.
Lane 1 – 100bp ladder
Lane 2 – gDNA (5/13/2009)
Lane 3 – gDNA (5/26/2009)
Lane 4 – H2O
Lane 5 – H2O
Lane 6 – H2O
Lane 7 – H2O
Results: Still contamination in the water-only samples!!!
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.
