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) Gel
 * 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

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




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