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Team:Warsaw/Calendar-Main/3 August 2009
From 2009.igem.org
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Cloning of p53 coding sequence
Marcin
Task 1:
- Isolation pSBA2 plasmid containing RFP CDS
Methods:
- Plasmids weren isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here.
Task 2:
- Clean-up the PCR product - amplified p53 CDS
Methods:
- DNA was purified using the A&A clean-up kit. Detailed procedure is described here
Task 2:
- Restriction digest of insert and vector sequence.
Comment
I decide that p53 coding sequence will be cloned to plasmid which is compatible with the Biobrick standard. Used plasmid contains CDS of RFP. After digest with SpeI and XbaI the sequence will be cut and the plasmid may be ligated with p53 which also will be digested using aforementioned enzymes. However the maximum effectiveness of the ligation is 50% because of the possibility of cloning the insert in opposite direction.
Methods:
- Digest for subsequent cloning using XbaI and SpeI
- Reaction mixture composition: 25 μl purified PCR product (or, 10 μl plasmid), 1 μl XbaI (Fermentas), 1 μl SpeI (Fermentas), 5 μl Buffer Tango (Fermentas), required μl MQ water to reach final volume of 50 μl
- Both reaction were perform in the same condition:
Program:
digest:
1. 37°C - 6 hours 2. 80°C - 15 minutes 3. 4°C - hold
- Purification of digested products via gel-out
Procedure:
- Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described here
- Quantification of amount of p53 DNA after restriction digest:
1 μl of the digest mixture was diluted to 10 μl and loaded into the gel.
Task 3:
- Cloning p53 coding sequence to the vector
Methods:
- Ligation mixture composition: 11 μl digested p53, 11 μl digested pKS, 2.5 μl ligation buffer (Fermentas), 1 μl ligase T4 (Fermentas)
- Duration of ligation was about 12 hours
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