Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/04/07

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry


Trying golden gate for receiver intermediate.
Not doing gel extraction and instead using DpnI digest and low concentration of plasmid in PCR.

No. Template Primer 1 Primer 2 Annealing Temp
1I13522-2 1:100P111P11262
2I13522-3 1:1000P111P11262
4I13522-2 1:100P103P10449
5I13522-3 1:1000P103P10449

Ran reactions 1,2,3 on Haynes lab PCR machine with annealing temp 61, 3.5 min extension time and 35 cycles.
Ran reactions 4 and 5 on Wang lab PCR machine with annealing temp 49, 1 min ext time and 35 cycles.

DpnI digest
Positive control was a miniprepped plasmid at 100ng/ul concentration. Incubated at 37°C for 40 minutes.

' Volume (ul) '
ThingPositive ctrlNegative ctrl
Green digest buffer22
pTet mCh55

Only digested reactions 1, 2, 4, and 5. Rxn 3 does not have plasmid DNA, the template is a g-block fragment.

Thing Volume (ul)
Digest buffer2
PCR rxn17

Heat inactivated for 5 minutes at 80°C about 15 minutes after taking them out of 37°C, forgot about that....
Ran gel of 10 ul of positive DpnI control (should only be cut up stuff, maybe a smear, maybe nothing), 10ul of negative DpnI control (should see uncut plasmid only), 5ul of PCR rxns 1-5 (not DpnI digested)

Personal tools