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) '
ThingPositive ctrlNegative ctrl
H2O1212
Green digest buffer22
pTet mCh55
DpnI10



Thing Volume (ul)
Digest buffer4
PCR rxn25
H2O1
DpnI1

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
1Rec bbGel extracted0.020.01220.289
2pLux-GFPDpnI digested, column purified0.0470.024247.148
3pRhl-GFPDpnI digested, column purified0.050.0252.00450.466
4pEsa-GFPDpnI digested, column purified0.0320.0152.11732.1
5pLac-LuxRDpnI digested, column purified0.0460.0241.93545.771
6pLac-RhlRDpnI digested, column purified0.060.0351.69259.88
7pLac-RhlRDpnI digested, column purified0.0410.0192.241.438
9pLac-EsaRDpnI digested, column purified0.0380.0182.13537.847

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.


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