Debugging IVT #

Yesterday used pFC11 and pFC8 for IVT experiments, but should have realized pFC11 is not R-loop forming. However, pFC8 did not show any transcription. I want to both retest a R-loop forming plasmid with a T7 promoter and 2 T3 R-loop formers to see if the T3 needs to be reordered.

The full FC plasmid table with respect to their promoters and R-loop forming abilities is below.

Currently have pFC8, 11, 14, 15, 17, 32 and 44 in the IVT box.

Also re-read the protocol and was using too much DTT (should be 10 mM) and way too much RNaseA. Fred says dilute 0.1mg/ml to 100x. That was probably eating up any R-loops that might have formed.

Protocol #

pFC8 DNA concentration: 240 ng / ul. pFC14 DNA concentration: 1452 ng / ul.

Diluted stock RNaseA 100x and added 0.5 ul per sample. Both samples use the T3 Pol.

Run gel for 2hr at 60v, afterwards stained with EtBr for ~15 mins but added 8 ul instead of the usual 2 ul.

Gel image #

Labeling scheme #

Reagent details #

After talking with Fred agreed there was some shift but a lot of noise, the source of which could be bad reagents. Checked all reagents and multiple were expired. Also noted lot numbers, but should order all new ones tomorrow.