Author Archives: kubu4

qPCRs – MV hemocyte cDNA from 20090614

qPCR – BgBL Primers

Set up qPCR with Cv_BgBL_F/R primers. This is a second rep. Plate layout/PCR set up is here.

Results: Waters are clean. To be analyzed later with other genes that Mac has run.

 

qPCR – HMG Primers

Set up qPCR with Cv_HMG_F/R primers. This is a second rep. Plate layout/PCR set up is here.

Results: Waters are clean. To be analyzed later with other genes that Mac has run.

 

qPCR – HSP70 Primers

Set up qPCR with Cv_HSP70_F/R primers. This is a second rep. Plate layout/PCR set up is here.

Results: Waters are clean. To be analyzed later with other genes that Mac has run.

qPCRs – MV hemocyte cDNA from 20090614

qPCR – CysB Primers

Set up qPCR with Cv_CysB_F/R. Plate layout/PCR set up is here.

Results: Waters are clean. To be analyzed later with other genes that Mac has run.

 

qPCR -BgBp Primers

Set up qPCR with Cv_BgBp_F/R primers. Plate layout/PCR set up is here.

Results: Waters are clean. To be analyzed later with other genes that Mac has run.

qPCR – MV hemocyte cDNA from 20090614

Set up qPCR with Cv_18s_F/R primers. Plate layout/PCR set up is here. This is a second rep. Used 1:20 cDNA plate made by Mac as template.

Results: Waters are clean. Still some samples that aren’t coming up. Will eventually run those samples using undiluted cDNA. To be analyzed later with other genes that Mac has run.

qPCR – Abalone Dg DNased RNA from yesterday and earlier today

Check Abalone RNA for residual gDNA contamination after DNase treatment. Ran qPCR with H.crach_16s_SYB_F/R primers. Plate layout/PCR set up here.

Results: Still gDNA in virtually every sample! This is totally insane. Will find/design primers that will only amplify gDNA to aid in subsequent analysis…

qPCRs – MV hemocyte cDNA from 20090614

qPCR – CIAPIN Primers

Set up qPCR with Cv_CIAPIN_F/R primers. Plate layout/PCR set up is here.

Results: Waters are clean. gDNA samples did not amplify. To be analyzed later with other genes that Mac has run.

 

qPCR – CatY Primers

Set up qPCR with Cv_CatY_F/R primers. Plate layout/PCR set up is here.

Results: Waters are clean. gDNA samples did not amplify. To be analyzed later with other genes that Mac has run.

EtOH Precipitation – DNased Abalone Dg RNA from yesterday AND the 07:12 set (DNased by Lisa 20090306)

Due to the excessive number of times that these samples have been DNased, I’m concerned that the buffer is becoming too concentrated. So, I’m performing an EtOH precip on them to clean them up and then proceed with another round of DNase treatment.

0.1 volumes of 3M sodium acetate (pH = 5.2) were added to each sample. Then, 2 volumes of 100%, ice-cold EtOH were added. Samples were mixed and stored @ -20C for 2hrs. Samples were then centrifuged @ 4C, 16,000g for 30mins. Supe removed. Added 1mL of 70% EtOH and then centrifuged samples @ 4C, 16,000g for 15mins. Supe was removed, tubes were spun briefly to pool residual EtOH and the remaining EtOH was removed. RNA was resuspended in 20uL of 0.1% DEPC-H2O and spec’d.

Results: The following samples appear to have no RNA after precipitation:

06:5-34, 37; 06:6-44, 45, 46, 47, 52; 08:4-9, 10, 13, 15-18; All of the 07:12 samples.

Since I did not DNase the 07:12 samples, I can’t say whether or not this result was expected (e.g. due to low initial concentration). The others that have no remaining RNA are a surprise and is a bit disconcerting.

Will take fresh RNA aliquots of those samples for DNasing. For those samples still having RNA post-recip, I will just use them as they are, as most concentrations are in the recommended range for DNasing, according to the Ambion Tubro DNA-free kit.