User:Howard Boland/Notebook/Art from Synthetic Biology/2010/11/04

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PCR Product, 2.3kb product
So, yesterday I finally got some bands back. I am choosing to work with the 25-10-10-BLK template as it had the more prominent bands. I am also adding more reagents to my Master mix to try to boost the yield as it is still invisible with the naked eye.

Master Mix
 * 1) 77µl H2O
 * 2) 8µl 10xPFU Buffer
 * 3) 3µl Forward Primer *1.5 per reaction rather than 1.25
 * 4) 3µl Reverse Primer *1.5 per reaction rather than 1.25
 * 5) 4µl Template
 * 6) 3µl dNTP *1.5 per reaction rather than 0.4
 * 7) 2µl PFU DNA Polymerase

Aliquote into each of 2 tubes
 * 1) 50µl Master Mix

PCR conditions

Cycles: 30x Lid: 100ºC Volume: 50µl (each)


 * 1) Initial: 94ºC, 1 min
 * 2) Denature: 94ºC, 30 sec
 * 3) Annealing (Tm): 55ºC, 50sec  * down from 56ºC
 * 4) Extension: 72ºC, 4min 45sec (2 min/kb x 2.359kb) * up from 4:37
 * 5) Goto 2, 30 times
 * 6) Final: 72ºC, 10 min
 * 7) Rest: 8ºC, forever

Gel

I prepared a 1% Agarose Gel
 * 1) Lane 1: 10µl  1kb NEB Quick Ladder
 * 2) Lane 2: BLANK
 * 3) Lane 3: 50µl  PCR product (2300bp) 25-10-10-BLK
 * 4) Lane 4: BLANK
 * 5) Lane 5: 50µl  PCR product (2300bp) 25-10-10-BLK
 * 6) Lane 6-8: BLANK

Error: Bands were not clear enough to be seen with naked eye. Lane 5 shows potential band result but unfortunately too weak to cut.

PCR Product, 2.3kb product
I prepared two separate PCR reaction for the same product amplification

Reactions
 * 1) 40.1µl H2O
 * 2) 4µl 10xPFU Buffer
 * 3) 1.25µl Forward Primer
 * 4) 1.25µl Reverse Primer
 * 5) 2µl Template
 * 6) 0.4µl dNTP
 * 7) 1µl PFU DNA Polymerase

PCR conditions

Cycles: 30x Lid: 100ºC Volume: 50µl (each)


 * 1) Initial: 94ºC, 1 min
 * 2) Denature: 94ºC, 30 sec
 * 3) Annealing (Tm): 56ºC, 50sec
 * 4) Extension: 72ºC, 4min 37sec (2 min/kb x 2.359kb)
 * 5) Goto 2, 30 times
 * 6) Final: 72ºC, 10 min
 * 7) Rest: 8ºC, forever

Ligation
I am retrying ligation of the previous purified products. I set up two reactions

Reaction
 * 1) 6µl pBR322-PstI-Purified, 790bp
 * 2) 2µl pSense66-PstI-Purified, 2300bp
 * 3) 1µl Ligase Buffer
 * 4) 1µl Ligase

PCR conditions

Lid: 100ºC Volume: 10µl (each)
 * 1) Rest: 14ºC, forever

The PCR reaction was run overnight


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