Chloroquine gel imaging and Gibson assembly reaction component gathering #

Chloroquine gel wash and imaging #

Arrived at the lab around 7:00 am and removed chloroquine gel from the gel box. Fire alarm went off at 7:15 and had to leave the lab. Chloroquine gel in npH20 wash for two hours. At 9:44 I removed the gel from the wash and post stained with EtBr at a concentration of 0.04 ul/ul. After post staining for 30 minutes I imaged the gel using the 2nd floor BioRad imager.

pFC9 shows the greatest degree of smearing, and the darkest bands indicating a higher concentration of DNA compared to the other samples. This may be due to the fact all samples except pFC9 are from mini-preps and may have some residual RNA. Various degrees of shifting are observable between the different plasmids. Fred did bring up the point though that we are not really learning anything new from this gel and that running a gel with R-loop containing plasmids that also has chloroquine in it may not really make sense as the effect chloroquine has on R-loop structures is not really known. At least now I should be able to consistently do this assay if need be but its utility for examining in greater detail the results of IVT as I hoped I would be able to do is in question and unlikely to be worth further pursuing.

BglII digestion followed by PCR amplification of vector bound VR inserts #

Digested samples VR 8, 12, 15, 16, 17, 20, 21, 23, 25, 27 and 31 with BglII to remove fragments from supplied vectors.

Digestion conditions #

Each digestion occurred in 10 ul total reaction volume (same reaction setup as preformed on 9/23/21).

• 10 ul total reaction volume
  • 0.2 ul BglII
  • 1 ul insert vector (50 ng)
  • 1 ul 10x r3.1 buffer
  • 7.8 ul npH20 (opened 9/27/21)

I also included the following negative controls.

• C: H20 + BglII buffer
• CB: C + BglII
• CBPCR: CB + PCR master mix

All controls were PCR amplified along with vector samples. All samples were incubated in thermocycler at 37C for 1 hour. Digest completed at 9:15 AM. I then placed all samples into deli fridge. At 10 AM I prepped a PCR master mix to amplify the inserts in hood in the tissue culture room and add 25 ul PCR master mix to each BglII digestion.

Digestion and PCR results #

After amplification ran PCR products on gel. Loaded 3ul each sample with 3 ul purple loading dye and 7 ul H20 onto 0.8% agarose gel made with 1x TAE in a 1x TAE buffer with 2 ul/100ml EtBr in both buffer and agarose. I then ran the gel at 120V for 45 minutes.

Glass bead cleaning #

Cleaned glass beads for E. coli transformations. Soaked beads in 10% bleach npH2O solution for 20 mins starting at 8:48 am. After rinsing I placed beads in oven to dry overnight.