qPCR – DNased MV hemocyte RNA from earlier today AND Turbo kit test

qPCR set up/plate layout is here. Used Cv_18s_F/R primers for the MV hemocyte RNA and Gigas_18s_F/R primers for the Turbo kit test. Anneal 55C.

Results: DNase treatment worked on all but the following samples: B23, B14, A21. However, these three samples were slightly below the initial, background fluorescence in each sample. The Turbo kit test indicates that all three kits are working perfectly and all can/should be used with confidence for treating samples.

DNase Treatment – MV hemocyte RNA from yesterday

Samples 3326: B23, A25, A22, B14, A21, A10 B22 came up positive for gDNA still. These were retreated according to Ambion protocol with a brand new Turbo DNA-free DNase kit. Additionally, I tested all three existing kits by “spiking” 19uL of H2O with 1uL (~200ng) of gigas gDNA; one tube for each kit and an untreated sample. Will qPCR to see if gDNA removal was successful.

DNase Treatment – MV hemocyte RNA from earlier today

The entire 20uL of RNA were treated with Ambion’s Turbo DNA-free kit according to protocol and spec’d.

Mac performed qPCR on the DNase-treated RNAs to verify removal of residual gDNA.

Results: Samples 3326: B23, A25, A22, B14, A21, A10 B22 came up positive for residual gDNA. Will retreat these samples and qPCR again.

RNA Isolation – Martha’s Vineyard (MV) hemocytes

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:

  • A7
  • A10
  • A15
  • A17
  • A20
  • A22
  • A26
  • 3326:
  • B1
  • B8
  • B10
  • B13
  • B14
  • B22
  • B26
  • B29

RNAs were resuspended in 10uL of 0.1% DEPC-H2O. They will be DNase treated.

qPCR – Re-DNased abalone Dg RNA from earlier today

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.

qPCR – Re-DNased abalone Dg RNA from earlier today

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.

PCR – C.pugetti gDNA from 20090526

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…

PCR – C.pugetti gDNA from 20090513 & 20090526

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!!!