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

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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.