Template:SJTU-BioX-Shanghai/Notebook/Construction/w4

Construction

July 29

1. Overlapping PCR for the whole construction of P_dark-HR* and pCPCG2-HR* using new primers in order to add new restriction enzyme reaction sites XbaI and PstI.

   	P_dark-HR*	pCPCG2-HR*
   ddwater	88μl	88μl
   Template1	P_dark-HR-PcpcB 1.3μl(49.7ng/50μl)	pCPCG2-HR-PcpcB 1.4μl(52.8ng/50μl)
   Template2	PcpcB-Kn 2.9μl(55.7ng/50μl)	PcpcB -Kn 2.9μl(55.7ng/50μl)
   Primer1	4μl	4μl
   Primer2	4μl	4μl
   PrimeSTAR Mix	100μl	100μl
   Total volume	200μl(25μl*8)	200μl(25μl*8)
   2-step PCR （round 1）×8 cycle	98℃ 3min
   	98℃ 10s
   	72℃ 1min15s
   	72℃ 3min
   	12℃ ∞
   2-step PCR (round2) ×33 cycle	98℃ 3min
   	98℃ 10s
   	72℃ 1min50s
   	72℃ 3min
   	12℃ ∞
   Final concentration (after purification)	72.4ng/μl	67.7ng/μl

   Gel analyses and purification/gel extraction.

July 30

1. Restriction reaction and ligation reaction of pBluescript and DNA fragments P_dark-HR*, pCPCG2-HR* with XbaI and PstI.

   Digestion reaction	pBluescript	P_dark-HR*	pCPCG2-HR*
   ddwater	45μl	86.8μl	90μl
   10×(green) buffer	10μl	8μl	8μl
   Plasmid/DNA fragment	35μl(1μg)	17.2μl(1μg)	14μl
   XbaI	5μl	4μl	4μl
   PstI	5μl	4μl	4μl
   Total volume	100μl(20μl*5)	120μl(30μl*4)	120μl(30μl*4)
   Reaction	37℃ for 20min	37℃ for 1h	37℃ for 1h
   Denaturation	Gel extraction	Purification	Purification

   
   

   Ligation reaction	P_dark-HR*-pBluescript	pCPCG2-HR*-pBluescript
   ddwater	2.8μl	2.8μl
   10×buffer	1μl	1μl
   Vector DNA	1μl(72.1ng)	1μl(72.1ng)
   Insert DNA	5μl(362ng)	5μl(338.5ng)
   Ligase	0.3μl	0.3μl
   Total volume	10μl	10μl
   Reaction	17℃ for 4h	17℃ for 4h
   Denaturation	Thermodynamic	Thermodynamic

2. Transformation of P_dark-HR*-pBluescript and pCPCG2-HR*-pBluescript.

July 31

1. Inoculation: 16 samples for each construction.

Aug. 1

1. Bacterial liquid PCR.

   Forward primer	1μl (10μM)
   Reverse primer	1μl (10μM)
   Template(bacterial liquid)	1μl
   2×Taq Mix	10μl
   diwater	Fill up to 20μl
   Total volume	20μl
   ×25 cycle	95℃ 3min
   	94℃ 15s
   	68℃ 15s
   	72℃ 3min50s
   	72℃ 5min
   	12℃ ∞

   Gel analyses.

1. Plasmid extraction.
2. Restriction enzyme reaction examination.

1. Transformation of pCPCG2-HR*-pBluescript, using blue-white screen.
2. Prepare 10× TBE mother liquor.
3. Sequence the examined plasmid containing construction P_dark-HR*-pBluescript.

Aug. 2

1. Inoculation: 17 samples.
2. Bacterial liquid PCR.