User:Howard Boland/Notebook/Art from Synthetic Biology/2010/10/26

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Gel pUA66katE template, product pSense66 - 2359bp
Should have used 1% Gel, 1kb ladder - product loaded on wrong gel (gel mix-up)


 * 1) Lane1: 10µL 100bp NEB Quick Ladder
 * 2) Lane2: BLANK
 * 3) Lane3: 50µl PCR (pUA66katE expected 2359bp) Digested with PstI
 * 4) Lane5-8: BLANK



Gel pMAK512 template, product pBR322 - 790bp
Should have used 2% Gel, 100bp ladder - product loaded on wrong gel (gel mix-up)


 * 1) Lane 1: 10µL  1kb NEB Quick Ladder
 * 2) Lane 2: BLANK
 * 3) Lane 3: 50µl  PCR pBR322, digest PstI, Cut
 * 4) Lane 4: BLANK
 * 5) Lane 5: 5µL pUA66katE (Circular)
 * 6) Lane 6: 5µL pUA66katE (Circular)
 * 7) Lane 6: 5µL pMAK512 (Circular)
 * 8) Lane 7: 5µL pMAK512 (Circular)



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

Mastermix PCR - pMAK512
 * 1) 80.2µl H20
 * 2) 8µl 10xpfu Polymerase Buffer
 * 3) 2µl pMAK512 plasmid template (new template from 25-10-2010)
 * 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): 60ºC, 50sec (was 58ºC)
 * 4) Extension: 72ºC, 1min 15sec (was 1:35)
 * 5) Goto 2, 30 times
 * 6) Final: 72ºC, 10 min
 * 7) Rest: 8ºC, forever

Mastermix PCR - pUA66katE
 * 1) 80.2µl H20
 * 2) 8µl 10xpfu Polymerase Buffer
 * 3) 2µl pUA66katE plasmid template (new template from 25-10-2010)
 * 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): 58ºC, 50sec (was 56ºC)
 * 4) Extension: 72ºC, 4min 15sec (was 4:43)
 * 5) Goto 2, 30 times
 * 6) Final: 72ºC, 10 min
 * 7) Rest: 8ºC, forever

Possible error: There was a lot of kerfuffle around me whilst setting up these reactions

Gel purification
The last step I did was to purify products from the gels. Gels were however not accurate and I will need to check these purified products tomorrow on correct gels.

I purified pBR322 from fridays PCR and digestion (overnight and 2 hour) and one of the pSense66 products from the overnight digestion.


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