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


Image:26102010-2prcent.jpg

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)

Image:26102010-1prcent.jpg

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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