Tag Archives: Quantitect Kit

PCR – Test QT Kit with No RT Abalone rxns from 20090408

Anneal 55C. PCR set up is here (bottom half of sheet).

Lane 1 – Hyperladder

Lane 2 – gDNA

Lane 3 – cDNA pool

Lane 4 – No RT 06:5-31

Lane 5 – No RT 06:6-43

Lane 6 – No RT 08:3-5

Lane 7 – No RT 08:3-6

Lane 8 – No RT 08:3-7

Lane 9 – H2O

Lane 10 – H2O

Lane 11 – 100bp ladder

Results: No signal in the gDNA. Appropriate sized band in cDNA pool. Nothing in the water samples. HOWEVER, got bands in tow of the “No RT” rxns (08:3-6/7)!! It’s odd that the gDNA didn’t produce any signal, but there shouldn’t be any signal in the No RT rxns. This indicates gDNA contamination. Should have also tested RNA and DNased RNA. Will test these in order to determine if that system is better at eliminating gDNA carryover.

cDNA – Abalone RNA from 20090331 & 20090402

cDNA was made from the above RNA samples using the Qiagen Quantitect RT Kit. The samples were laid out in a PCR plate. 274.2ng of RNA was used in the rxn for each sample, based on the lowest concentration RNA sample (08:3-20) to equalize all the samples. The Genomic Wipeout step of the kit requires 2uL of Genomic Wipeout enzyme/buffer to be added to 12uL of an RNA sample, so the calculations were done and can be found here. A check mark on the calculation sheet indicates that the water and then the RNA was added to the appropriate wells. Those with two check marks were used for a “No RT” rxn and thus, have duplicate wells (see plate layout).

The plate was mixed, spot spun, uncubated at 42C for 2mins and immediately placed on ice.

The RT and No RT master mixes were set up on ice and then added to the respective wells (see sheet here).

UPDATE: cDNA plate was discarded 20120320 by SJW.