Identifying contamination in BglII digestions and amplification #

Set up series of PCR reactions in order to identify reagent or reagent(s) that may contain the contaminate identified on 9-21-21.

BglII reagent screen #

Set up PCR using same master mix I used in 9-21-21 digestions with individual digest reagents shown in the table below.

After 2 hour PCR reaction ODed each sample results of which are below.

All samples showed amplification and no insert was added to each sample. The sample master mix was used for each sample so it seems likely that one or more of the reagents that compose the PCR master mix is contaminated.

PCR master mix reagent screen #

Since PCR is required for determine if contamination is present I set up reactions where one of the reagents I used to create the offending master mix was absent. Reactions that do no show amplification when a reagent is swapped out will reveal the contaminating reagent.

Sample 4 was the PCR master mix itself. Added VR insert primers to each sample and then ran 2 hour PCR reaction.

From this test it looks like the dNTPs have been contaminated with something.

PCR grid test #

Set up many reactions to test as many combinations of reagents as possible.

All reactions set up in 10 ul volumes in Colin’s lab PCR tubes. Ran 34 cycles standard PCR reaction. Left samples overnight to OD and run some on gel the next morning.