(Difference between revisions)
Line 22: Line 22:
<div id="nav">
<div id="nav">
<ul class="dropdown dropdown-horizontal">
<ul class="dropdown dropdown-horizontal" style="background: none;">
  <li><a href="" class="dir">home</a>
  <li><a href="" class="dir">home</a>

Revision as of 09:51, 6 October 2010


Repetitive failure of purifications with both traditional method and kits, unsure what is going wrong. Have decided to tell Chris. Off to Firbush on Wednesday.

In my absence (at Firbush with the rest of the Biotechnologists) Chris has managed to construct the tnaA UP-cat-sacB-tnaA DOWN segment that we need for the first part of the BRIDGE protocol. I will continue to build the tnaA UP-RFP-tnaA DOWN segment.
Set up ligations of RFP-DOWN and UP-RFP, left in 16C incubator overnight.

Lab on a Saturday again, fun fun fun...
Retrieved and amplified ligations and ran on gel. UP-RFP appears to be significantly shorter than RFP-DOWN and indeed shorter than it ought to be (should be about 2kb, is only around 1.5kb).
Purified both products, then digested RFP-DOWN with XbaI. (Used 2hour incubation time to write presentation for Wednesday).
Set up ligation of UP with RFP-DOWN and left in incubator overnight.

Bus to Darwin for 11:20am; enter Darwin; lift to 8th floor; retrieve ligations from waterbath in cold room; walk down to 7th floor; put ligation in freezer; lift to ground floor; leave Darwin; catch 11:40am bus back to central.

PCR of ligation of UP-RFP-DOWN. Made and ran gel along with purified PCRs from Saturday, just in case they're still not working.
They worked! The purifications were perfect. There is a band about the size of UP-RFP-DOWN in it's lane, but it's quite faint. I will probably repeat the PCR with a lower annealing temperature (around 50) this afternoon. I suspect the UP primers do not anneal properly above 54C.