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

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04/07/2014

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

No. Template Primer 1 Primer 2 Annealing Temp
1 I13522-2 1:100 P111 P112 62
2 I13522-3 1:1000 P111 P112 62
3 G001 P105 P107 61
4 I13522-2 1:100 P103 P104 49
5 I13522-3 1:1000 P103 P104 49


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) '
Thing Positive ctrl Negative ctrl
H2O 12 12
Green digest buffer 2 2
pTet mCh 5 5
DpnI 1 0

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 buffer 2
PCR rxn 17
DpnI 1

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)