User:Howard Boland/Notebook/Art from Synthetic Biology/2010/10/27

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

2% Gel PCR pBR322 (790bp)
Using the new optimised conditions I ran a 2% Gel to check if I got the pBR322 product and what background noise yield it produced.


 * 1) Lane 1: 100bp NEB Quick Ladder
 * 2) Lane 2: Blank
 * 3) Lane 3: 50µl PCR Product pBR322, expected 790bp
 * 4) Lane 4: Blank
 * 5) Lane 5: 50µl PCR Product pBR322, expected 790bp
 * 6) Lane 6: Blank
 * 7) Lane 7: 5µl pBR322-PstI-purified
 * 8) Lane 8: 5µl pBR322-PstI-purified

Gel Documentation

1% Gel PCR pSense66 (2302bp)
Using the new optimised conditions I ran a 1% Gel to check if I got the pSense66 product and what background noise yield it produced.


 * 1) Lane 1: 1kb NEB Quick Ladder
 * 2) Lane 2: Blank
 * 3) Lane 3: 50µl PCR Product pSense66, expected 2302bp
 * 4) Lane 4: Blank
 * 5) Lane 5: 50µl PCR Product pSense66, expected 2302bp
 * 6) Lane 6: Blank
 * 7) Lane 7: Blank
 * 8) Lane 8: 5µl pSense-PstI-purified

Gel Documentation

Ligation
I setup a 10μl ligation between the 700bp and the 2300bp product.


 * 1) 0.5μl Ligase
 * 2) 1μl Ligase Buffer
 * 3) 2μl pSense66 (2300bp)
 * 4) 3μl pBR322 (790bp)
 * 5) 4.5μl H2O

Incubate for 37°C for 10 minutes.

Transformation
The ligated product was heat-shock transformed and I also transformed 1μL of the sequenced pUA66katE plasmid.

PCR Optimisation
This protocol is a review to correct the PCR conditions from Monday.

Mastermix PCR - pUA66katE
 * 1) 80.2µl H20
 * 2) 8µl 10xpfu Polymerase Buffer
 * 3) 2µl pUA66katE plasmid template (sequenced template)
 * 4) 2.5µl Primer forward
 * 5) 2.5µl Primer reverse

For each reaction add the following to each 50µl PCR tube
 * 1) 47.6µl Mastermix
 * 2) 0.4µl 10mM dNTP (not supplied with kit)
 * 3) 1µl pfu Polymerase

PCR conditions

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


 * 1) Initial: 94ºC, 1 min
 * 2) Denature: 94ºC, 30 sec
 * 3) Annealing (Tm): 57ºC, 50sec (was 58ºC)
 * 4) Extension: 72ºC, 4min 30sec (was 4:15)
 * 5) Goto 2, 30 times
 * 6) Final: 72ºC, 10 min
 * 7) Rest: 8ºC, forever


 * }