Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/04/29

Project name

Main project page Previous entry      Next entry

mm/dd/yyyy Ran PCR products from yesterday on a gel. 5ul of reactions 2-9, all 30 of 1 so I can gel extract because there should be two bands and I only want one. DpnI digest PCR reactions 2-7 and 9 with positive and negative controls (didn't run reaction 8 because I checked the gel early and saw that there was no DNA there). Controls Positive control was a miniprepped plasmid at 100ng/ul concentration. ' Volume (ul) ' Thing Positive ctrl Negative ctrl H2O 12 12 Green digest buffer 2 2 pTet mCh 5 5 DpnI 1 0 Thing Volume (ul) Digest buffer 4 PCR rxn 25 H2O 1 DpnI 1 Incubated at 37°C for 55 minutes, sat on the bench for 3 minutes, heat inactivated at 80 for 5 minutes. Cleaned using zymo DNA clean and concentration-5 kit (columns from gel extraction kit). Added 150ul of DNA binding buffer to DpnI reactions. Gel extracted PCR reaction 1. Saw a smaller band above, think I got the correct band. PCR reaction Description ' 260 280 260/280 ng/µL 1 Rec bb Gel extracted 0.02 0.01 2 20.289 2 pLux-GFP DpnI digested, column purified 0.047 0.024 2 47.148 3 pRhl-GFP DpnI digested, column purified 0.05 0.025 2.004 50.466 4 pEsa-GFP DpnI digested, column purified 0.032 0.015 2.117 32.1 5 pLac-LuxR DpnI digested, column purified 0.046 0.024 1.935 45.771 6 pLac-RhlR DpnI digested, column purified 0.06 0.035 1.692 59.88 7 pLac-RhlR DpnI digested, column purified 0.041 0.019 2.2 41.438 9 pLac-EsaR DpnI digested, column purified 0.038 0.018 2.135 37.847 Didn't have enough of the backbone so started another PCR. 1-4 are int receiver vector P112 P123, 5-6 are purified PCR product of receiver backbone P112 P123.