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

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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 Image:27102010-2prcnt-gel-pBR322-pcr.jpg

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 Image:27102010-1prcnt-gel-pSense66-pcr.jpg

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



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