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

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==04/07/2014==
==04/07/2014==
Trying golden gate for receiver intermediate. <br>
Trying golden gate for receiver intermediate. <br>
***Changes*** <br>
'''Changes''' <br>
Not doing gel extraction and instead using DpnI digest and low concentration of plasmid in PCR.
Not doing gel extraction and instead using DpnI digest and low concentration of plasmid in PCR.
<br>
<br>

Revision as of 11:36, 22 April 2014

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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)



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