Author Archives: kubu4

gDNA Isolation – Gigas Dermo Samples

Transferred tissue from previously Chelexed samples:

WP3, WP7, WP13, WP14, WP15, CY1 – These all tested negative for virginica gDNA (18s qPCR by Rony on DATE)

CY42, CY36 – These tested positive for virginica gDNA (18s qPCR by Rony on DATE).

Used Qiagen DNeasy Kit. Digested samples for 3 hrs. at 55C in Proteinase K according to protocol.

Results: Horrible. Virtually no DNA in any of these samples. Additionally, the quality of the DNA is atrocious. Not sure what to do now.

gDNA Isolation – Mac’s BB and DH site samples

Due to failure of gDNA isolation via the TriReagent method (see 20090511) used Qiagen DNeasy Kit. Digested samples for 3 hrs. at 55C in Proteinase K according to protocol. Performed on a subset of each site samples: BB#11-18 & DH#11-18.

Results: Excellent yields and superb quality.

Reverse Transcription – Mac’s gigas DNased RNA from 20090512

Performed RT using Promega M-MLV RT according to M-MLV protocol and used 0.5ug oligo dT per ug of RNA on all BB and DH site samples that were negative for gDNA (see qPCR results 20090512). Calculations and work up are here. Samples were set up in a plate to facilitate sample loading in subsequent qPCRs.

UPDATE: cDNA plate was discarded 20120320 by SJW.

gDNA Isolation – C.pugetti

Isolated according to JGI protocol (Word doc). Used 100mL, 8 day old culture inoculated from a plate on 20090505. Resuspended pellets in 740uL of TE and took an OD600 via the NanoDrop. Diluted the sample appropriately to an OD600 ~ = 1.0 in a final volume of 740uL TE (see the last three measurements for OD600 of final dilution).

Followed protocol. Recovered 300uL of aqueous phase prior to precipitation with isopropanol (Step #21). Resuspended DNA in 20uL of H2O. Will run samples on gel according to JGI instructions.

Lane 1 – 15ng standard (5uL)

Lane 2 – 31ng standard (5uL)

Lane 3 – 63ng standard (5uL)

Lane 4 – Marker 2 (5uL)

Lane 5 – C. pugetti DNA (5uL: 3uL + 2uL dye)

Lane 6 – Marker 3 (5uL)

Lane 7 – 125ng standard

Lane 8 – 250ng standard (5uL)

Lane 9 – 500ng standard (5uL)

Results: DNA looks stellar! Just like the example gel in the JGI QC documentation. however, looks to be too little TOTAL yield of DNA to send for sequencing (need

qPCR – Mac’s gigas DNased RNA from earlier today

Performed qPCR on the DNased oyster RNA from earlier today with Gigas_18s_F/R primers to verify removal of detectable gDNA in the samples, since the qPCR from 20090508 indicated residual gDNA was still present in some of the DNase treated RNA. Plate layout/set up is here.

Results: Samples BB# 2, 5, 6, 15 still came up as positive for gDNA contamination. These will NOT be used to make cDNA for subsequent qPCRs.

DNase Treatment (Rigorous!) – Mac’s gigas RNA/Re-DNased RNA from 20090507 & 20090508, respectively

Followed the rigorous protocol for Ambion’s Turbo DNA-free protocol for the following RNAs:

BB#11-20

DH#11-20

Used 10ug of RNA (200ng/uL) in 50uL as directed. Here are the calcs for FF and DH.

Followed standard protocol on DNased samples from 20090508:

BB#1-10

DH#1-10

The standard protocol should be fine for these samples, since the procedure worked on Friday for some of them.

DNA Isolation – Mac’s gigas samples from 20090505 & 20090506

Isolated gDNA according to Molecular Research Center TriReagent protocol from BB#1-20 and DH#1-20. Resuspended DNA in 600uL of 8mM NaOH. Spec.

Results: HORRIBLE! This is some of the worst “DNA” I’ve ever seen. Peaks everywhere EXCEPT at 260nm. Here’s a link to the actual numbers. It’s a text file and is comma separated, so you should open with Excel for it to be readable.

Spoke with Steven. Will pursue RNA instead of continuing down this path for now.

qPCR – Re-DNased oyster RNA from today

Performed qPCR on the re-DNased oyster RNA from earlier today with Gigas_18s_F/R primers to verify removal of detectable gDNA in the samples, since the initial qPCR from yesterday indicated residual gDNA was still present in the DNase treated RNA. Plate layout/set up can be found here.

Results: About 4 samples in each site set are NEGATIVE for gDNA. That means the remainder still have detectable levels of gDNA. Boo.

DNase Treatment – Oyster RNA from yesterday

Yesterday’s qPCR indicated that all of the RNA still contained gDNA contamination. So, took 10ug of RNA from BB #1-10 and DH#1-10 (calcs/workup BB and DH) and brought the volumes up to 50uL with 0.1% DEPC-H2O. Processed the samples according to Ambion Tubrbo DNA-free protocol. Will proceed with qPCR immediately.