PCR Product, 505 bp product
Generate insert using colony PCR on 2 tubes each of 41µl volume
Colony Dilution
- Add 10µl water to an Eppendorf tube
- Add one colony E.coli <- ENSURE THAT SUFFICIENT COLONY IS TRANSFERED
- Mix well
Master mix (2x)
For a final volume of 70µl (2x35µl):
- 50µl Water
- 10µl 10xOptimized DyNAzyme Ext Buffer
- 2µl 10mM dNTP
- 4µl Primer: BBa_HB_katE_F (Forward)
- 4µl Primer: BBa_HB_katE_R (Reverse)
Reaction
- Aliquot 35µl Master Mix into each of two 0.5ml PCR tube
- Add 1µl Template DNA from the Colony Dilution
- For each tube add 5µl of DyNAzyme EXT DNA Polymerase
- 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)
- 94ºC for 05:00
- 94ºC for 00:30
- 52.5ºC for 00:20
- 72ºC for 10:00
- GOTO step 2, 32 times
- 72ºC for 10:00
- 8ºC forever
- END
Gel
I prepared a 2% Agarose Gel
- Lane 1: 10µl 1kb NEB Quick Ladder <= Error (Not sure why this happend as I check it)
- Lane 2: BLANK
- Lane 3: 41µl PCR product (505bp)
- Lane 4: BLANK
- Lane 5: BLANK
- Lane 6: 41µl PCR product (505bp)
- Lane 7-8: BLANK
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