User:Howard Boland/Notebook/Art from Synthetic Biology/2011/01/26

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PCR Product, 505 bp product

Generate insert using colony PCR on 2 tubes each of 41µl volume

Colony Dilution

  1. Add 10µl water to an Eppendorf tube
  2. Add one colony E.coli <- ENSURE THAT SUFFICIENT COLONY IS TRANSFERED
  3. Mix well

Master mix (2x)

For a final volume of 70µl (2x35µl):

  1. 50µl Water
  2. 10µl 10xOptimized DyNAzyme Ext Buffer
  3. 2µl 10mM dNTP
  4. 4µl Primer: BBa_HB_katE_F (Forward)
  5. 4µl Primer: BBa_HB_katE_R (Reverse)

Reaction

  1. Aliquot 35µl Master Mix into each of two 0.5ml PCR tube
  2. Add 1µl Template DNA from the Colony Dilution
  3. For each tube add 5µl of DyNAzyme EXT DNA Polymerase
  4. Place in PCR thermocycler

Reaction Environment

Tm = 57.5 = > 57.5-5 = 52.5

505bp => 0.505kp*(40s/1kb) = 20.2 sec

Lid: 100ºC; Volume: 41µl (each)

  1. 94ºC for 05:00
  2. 94ºC for 00:30
  3. 52.5ºC for 00:20
  4. 72ºC for 10:00
  5. GOTO step 2, 32 times
  6. 72ºC for 10:00
  7. 8ºC forever
  8. END

Gel

I prepared a 2% Agarose Gel

  1. Lane 1: 10µl 1kb NEB Quick Ladder <= Error (Not sure why this happend as I check it)
  2. Lane 2: BLANK
  3. Lane 3: 41µl PCR product (505bp)
  4. Lane 4: BLANK
  5. Lane 5: BLANK
  6. Lane 6: 41µl PCR product (505bp)
  7. Lane 7-8: BLANK

28012011-pcr-bba kate.jpg