Wed, Sep 29 21

Chloroquine gel round two results #

Picked up chloroquine gel I started yesterday. Prepared gel for imaging as described on 9/28/21.

Lane layout #

pFC9 substrate.

2+ Topo
3+ Topo + Gyrase

Gel image #

Need to get off flashdrive.

Looks like the DNA has run off the gel. Need to adjust running time. Maybe previously we had some kind of mega gel?

PCR contamination test round three #

Set up four samples with same master mix I used in 9/28/21 PCR tests but with a Taq I purchased today from the scientific store (never been touched before) which also came with two 10X buffer solutions.

ItemProduct codeLotCount
DNA Taq PolymeraseM0273S10099911
10x DNA Pol bufferB90145101145452

PCR results #

Lane layout #

11kb ladder
2Master mix
3Master mix
4Master mix + new taq
5MAster mix + new taq

Gel image #

Need to export image to jpg

Lanes with Taq show some contamination plus a possible additional band? This means that contamination is still spreading or one or more of the reagents I used in the master mix is not as clean as I thought it might be. Need to use reagents that I have never even seen before in my life.

New PCR mix, working in cell culture hood #

Working in the cell culture hood with a new set of pipettes a made a new master mix and dNTP mix which are described in the tables below.

dNTP mix #

dNTPs40 ul (*4)
npH20 fresh460 ul

Master mix (A) #

ReagentVolume (ul)
10x Buffer (Lot: 10114545 )30
H20 fresh103

From this master mix I fractioned 70 ul and added 2.5 ul of each VR insert primer. From this second fraction (B) I fractioned 35 ul and added 1 ul of Taq Pol (Lot: 1009991).

PCR reactions and gel #

Using the reagents described above I set up the following PCR reactions and ran each for 34 cycles using standard PCR reaction and left overnight

4A + VR23 Vector
5B + VR 23 Vector
6C + VR 23 Vector
7A + Taq