Tue, Sep 28 21

Chloroquine gel post staining and imaging #

Picked up gels that I started yesterday. Washed each in ~500 ul of npH20 for two hours with shaking. After I drained H20 and poststained with 200 ul 1 ul / ml EtBr for 45 minutes. After post stained rinsed for 5 mins with EtBr.

Gel layout #

14Topo + Gyrase
24Topo + Gyrase

Gel images #

DNA is not present on the gel. Looking back at my calculations I used way too much chloroquine in the gel. Maybe this caused bands to run backwards?

Made stock solution of chloroquine with 125 ug / ul and prepared new Gyrase activity assay as described in 9/27/21 notes and prepared chloroquine gel that actually follows the correct protocol using 2 ml of stock chloroquine solution in 98 ul agarose. Loaded samples and ran gel at 110V for 16 hours.

PCR contamination test #

Testing new PCR master mix with various Taqs. Using reagents I have never used before to test for Taq contamination (in theory).

Master mix #

5x GoTaq28522702
NEB dNTP0561010
H20NA Fresh

Polymerases #

Pol nameLot
Phusion Pol00783326
Lab taqNA (A on tube)

Made four reactions one with each polymerase and one negative control with only master mix. Amplfied all samples for 34 cycles using standard PCR protocol.

Gel #

Ran products on 0.8% agarose TAE gel with 1ul/ml EtBr in 1x TAE buffer at 120V for 45 mins.

1MM + Phu
2MM + Deepvent
3MM + lab taq
51kb ladder

Clearly contamination present in the Deepvent (lane 2) and lab taq (lane 3) lanes. Not sure what is going on with Phu that is producing a smear but Megan saw this in an unrelated test that used phusion but with no substrate (as is the condition here).

Need to get even more extreme in contamination battle.