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wiki:documentation:wiki:documentation:jun:19-03-2020_pcr_odr-1_promoter [2020/03/20 11:23] (current)
jliu created
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 +====== 19-03-2020 PCR odr-1 promoter ======
 +
 +Purpose of experiment: To amplify the odr-1 promoter using oLJ12/13 (1.7kb) and oLJ12/14 (0.7kb) \\  ​
 +Another fold of the purpose is to see whether there is something wrong with reagents/​machine related instead of the parameters. The odr-1p is much shorter and in theory easier to amplify than the ceh-34p. in addition, I will also use Ian Chin-Sang’s plasmid (odr-1p::​dsred) as another control. \\  ​
 +
 +A: use N2 lysate as template, oLJ12/13 \\  ​
 +B: use N2 lysate as template, oLJ12/14 \\  ​
 +C: use odr-1p::​dsred as template oLJ12/13 (1:50 dilution for plasmid template) \\  ​
 +
 +template DNA 2 \\  ​
 +5X phusion HF buffer 4 \\  ​
 +10mM dNTPs 0.4 \\  ​
 +10uM Fwd primer 1 \\  ​
 +10uM Rev primer 1 \\  ​
 +Phusion DNA polymerate 0.2 \\  ​
 +H2O 11.4 \\  ​
 +Total 20ul \\  ​
 +
 +Make 10X master mix and set up 8 tubes in gradient PCR. See below. \\  ​
 +
 +98C 30sec  \\  ​
 +
 +98C 10sec \\  ​
 +72C-63 20sec \\  ​
 +72C 25sec \\  ​
 +Do this for 30 cycles \\  ​
 +
 +72C 10min \\  ​
 +
 +Result: no band at all. \\  ​
 +
 +
 +
 +
  
wiki/documentation/wiki/documentation/jun/19-03-2020_pcr_odr-1_promoter.txt · Last modified: 2020/03/20 11:23 by jliu

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