Gel pUA66katE template, product pSense66 - 2359bp
Should have used 1% Gel, 1kb ladder - product loaded on wrong gel (gel mix-up)
- Lane1: 10µL 100bp NEB Quick Ladder
- Lane2: BLANK
- Lane3: 50µl PCR (pUA66katE expected 2359bp) Digested with PstI
- Lane5-8: BLANK
Gel pMAK512 template, product pBR322 - 790bp
Should have used 2% Gel, 100bp ladder - product loaded on wrong gel (gel mix-up)
- Lane 1: 10µL 1kb NEB Quick Ladder
- Lane 2: BLANK
- Lane 3: 50µl PCR pBR322, digest PstI, Cut
- Lane 4: BLANK
- Lane 5: 5µL pUA66katE (Circular)
- Lane 6: 5µL pUA66katE (Circular)
- Lane 6: 5µL pMAK512 (Circular)
- Lane 7: 5µL pMAK512 (Circular)
PCR Optimisation
This protocol is a review to correct the PCR conditions from Friday.
Mastermix PCR - pMAK512
- 80.2µl H20
- 8µl 10xpfu Polymerase Buffer
- 2µl pMAK512 plasmid template (new template from 25-10-2010)
- 2.5µl Primer forward
- 2.5µl Primer reverse
For each reaction add the following to each 50µl PCR tube
- 47.6µl Mastermix
- 0.4µl 10mM dNTP (not supplied with kit)
- 1µl pfu Polymerase
PCR conditions
Cycles: 30x
Lid: 100ºC
Volume: 50µl (each)
- Initial: 94ºC, 1 min
- Denature: 94ºC, 30 sec
- Annealing (Tm): 60ºC, 50sec (was 58ºC)
- Extension: 72ºC, 1min 15sec (was 1:35)
- Goto 2, 30 times
- Final: 72ºC, 10 min
- Rest: 8ºC, forever
Mastermix PCR - pUA66katE
- 80.2µl H20
- 8µl 10xpfu Polymerase Buffer
- 2µl pUA66katE plasmid template (new template from 25-10-2010)
- 2.5µl Primer forward
- 2.5µl Primer reverse
For each reaction add the following to each 50µl PCR tube
- 47.6µl Mastermix
- 0.4µl 10mM dNTP (not supplied with kit)
- 1µl pfu Polymerase
PCR conditions
Cycles: 30x
Lid: 100ºC
Volume: 50µl (each)
- Initial: 94ºC, 1 min
- Denature: 94ºC, 30 sec
- Annealing (Tm): 58ºC, 50sec (was 56ºC)
- Extension: 72ºC, 4min 15sec (was 4:43)
- Goto 2, 30 times
- Final: 72ºC, 10 min
- 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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