User:Howard Boland/Notebook/Art from Synthetic Biology/2010/11/04

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PCR Product, 2.3kb product

So, yesterday I finally got some bands back. I am choosing to work with the 25-10-10-BLK template as it had the more prominent bands. I am also adding more reagents to my Master mix to try to boost the yield as it is still invisible with the naked eye.

Master Mix

  1. 77µl H2O
  2. 8µl 10xPFU Buffer
  3. 3µl Forward Primer *1.5 per reaction rather than 1.25
  4. 3µl Reverse Primer *1.5 per reaction rather than 1.25
  5. 4µl Template
  6. 3µl dNTP *1.5 per reaction rather than 0.4
  7. 2µl PFU DNA Polymerase

Aliquote into each of 2 tubes

  1. 50µl Master Mix


PCR conditions

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

  1. Initial: 94ºC, 1 min
  2. Denature: 94ºC, 30 sec
  3. Annealing (Tm): 55ºC, 50sec * down from 56ºC
  4. Extension: 72ºC, 4min 45sec (2 min/kb x 2.359kb) * up from 4:37
  5. Goto 2, 30 times
  6. Final: 72ºC, 10 min
  7. Rest: 8ºC, forever


Gel

I prepared a 1% Agarose Gel

  1. Lane 1: 10µl 1kb NEB Quick Ladder
  2. Lane 2: BLANK
  3. Lane 3: 50µl PCR product (2300bp) 25-10-10-BLK
  4. Lane 4: BLANK
  5. Lane 5: 50µl PCR product (2300bp) 25-10-10-BLK
  6. Lane 6-8: BLANK

Error: Bands were not clear enough to be seen with naked eye. Lane 5 shows potential band result but unfortunately too weak to cut.


PCR Product, 2.3kb product

I prepared two separate PCR reaction for the same product amplification

Reactions

  1. 40.1µl H2O
  2. 4µl 10xPFU Buffer
  3. 1.25µl Forward Primer
  4. 1.25µl Reverse Primer
  5. 2µl Template
  6. 0.4µl dNTP
  7. 1µl PFU DNA Polymerase


PCR conditions

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

  1. Initial: 94ºC, 1 min
  2. Denature: 94ºC, 30 sec
  3. Annealing (Tm): 56ºC, 50sec
  4. Extension: 72ºC, 4min 37sec (2 min/kb x 2.359kb)
  5. Goto 2, 30 times
  6. Final: 72ºC, 10 min
  7. Rest: 8ºC, forever

Ligation

I am retrying ligation of the previous purified products. I set up two reactions

Reaction

  1. 6µl pBR322-PstI-Purified, 790bp
  2. 2µl pSense66-PstI-Purified, 2300bp
  3. 1µl Ligase Buffer
  4. 1µl Ligase

PCR conditions

Lid: 100ºC Volume: 10µl (each)

  1. Rest: 14ºC, forever

The PCR reaction was run overnight


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