User:Howard Boland/Notebook/Art from Synthetic Biology/2010/11/15

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PCR Product, 2.3kb product

I ran a repeat of previous PCR from 4th of November 2010.

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

Image:15112010-pcr-result.jpg

Purification of Gel products

After cutting the product, I purified it together with last weeks digested PCR product.

Digestion

At this point I am interested in whether or not the 2300bp products can be broken into fragments. According to the in sillico this should not be possible but the result of last weeks digestion indicated that this was possible. If my product remains at 2300bp then I am pretty certain that this is my product.

Master Mix

  1. 1.5µl 100xBSA
  2. 15µl 10xNEB#3
  3. 6µl PstI
  4. 37.5µl H2O

Total mix is 60µl ensure it is well mixed.

For each of 3 tubes add

  1. 20µl of Master Mix
  2. 30µl Purified PCR product (~2300bp)

Mix well and place in 37ºC for 2 hours

Gel Result of digested 2300bp PCR product

I am expecting the result of the digestion to resolve as one band only and additional faint band represents possible undigested bits. I ran the digestion on a 1% gel.

  1. Lane 1: 10µl 1kb NEB Quick Ladder
  2. Lane 2: Blank
  3. Lane 3: 50µl Digested PCR product, PstI (Cut)
  4. Lane 4: Blank
  5. Lane 5: 50µl Digested PCR Product, PstI
  6. Lane 6: Blank
  7. Lane 7: Blank
  8. Lane 8: 50µl Digested PCR Product, PstI (Older sample)

Gel Picture Image:15112010-pcr-digested-psti.jpg



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