User:Howard Boland/Notebook/Art from Synthetic Biology/2010/10/25

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Gel PCR Products(pUA66katE template, 2359bp)

After revising optimised condition. I ran the new PCR products on a 2% Gel - this should have been run on a 1% Gel.

  1. Lane 1: 1kb NEB Quick Ladder
  2. Lane 2: BLANK
  3. Lane 3: Undigested PCR Product, expected 2359bp (from pUA66katE - hereby named pSense66)
  4. Lane 4: BLANK
  5. Lane 5: Undigested PCR Product, expected 2359bp (from pUA66katE - hereby named pSense66)
  6. Lane 6: BLANK
  7. Lane 7: 5µL pMAK512 (template for the pBR322 pcr)
  8. Lane 8: 5µL pUA66katE (template for this pcr)

25102010-cut-2prcnt-gel-pUA66katE.jpg

Error: PCR product is not completely optimised

Gel PCR Products(pMAK512 template, 790bp, pBR322)

I ran the PCR products from the pMAK512 template on a 2% Gel - this should have been run on a 1% Gel.

  1. Lane 1: 1kb NEB Quick Ladder
  2. Lane 2: BLANK
  3. Lane 3: Undigested PCR Product, expected 790bp (from pMAK512 - hereby named pBR322)
  4. Lane 4: BLANK
  5. Lane 5: Undigested PCR Product, expected 790bp (from pMAK512 - hereby named pBR322)
  6. Lane 6-8: BLANK

25102010-CUT-1PRCNT-GEL-PMAK512.jpg

Error: Should have been run on a 2% Gel

PCR Product PstI Digestion

Master Mix:

  1. 10µl 10xNEB#3
  2. 1µl 100xBSA
  3. 3µl PstI
  4. 26µl H2O

I used one tube of each of the PCR reactions (pSense66 and pBR322) to complete the reactions I set up the following Tube 1: Add 20µl Master mix to 30µl pBR322 PCR product Tube 2: Add 20µl Master mix to 30µl pSense66 PCR product

Digest for 2 hours at 37ºC

Gel PCR Products, Digest and Undigested

  1. Lane 1: 1kb NEB Quick Ladder
  2. Lane 2: Digested PCR Product with PstI, expected 2359bp (from pUA66katE - hereby named pSense66)
  3. Lane 3: BLANK
  4. Lane 4: Digested PCR Product with PstI, expected 790bp (from pMAK512 - hereby named pBR322)
  5. Lane 5: BLANK
  6. Lane 6: Undigested PCR Product, expected 2359bp (from pUA66katE - hereby named pSense66)
  7. Lane 7: BLANK
  8. Lane 8: Undigested PCR Product, expected 790bp (from pMAK512 - hereby named pBR322)

25102010-1PRCNT-GEL-PCR-DIGESTED-AND-UNDIGESTED.jpg


Overnight PCR Product PstI Digestion

Master Mix:

  1. 10µl 10xNEB#3
  2. 1µl 100xBSA
  3. 3µl PstI
  4. 16µl H2O

I used one tube of each of the PCR reactions (pSense66 and pBR322) to complete the reactions I set up the following Tube 1: Add 15µl Master mix to 25µl pBR322 PCR product Tube 2: Add 15µl Master mix to 25µl pSense66 PCR product

Digest overnight at 37ºC