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From 2010.igem.org

BRIDGE

Run gel:

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
Ladder	Pure SacB	Pure SacB & EcoRI	sacB & catR PCR	I15009 digest	S284T PCR Product	Purified 356K	Purified 356R

25/10/10
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.

01/10/10
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.

02/10/10
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.

03/10/10
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.

04/10/10
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.