Team:Warsaw/Calendar-Main/13 July 2009
From 2009.igem.org
Cloning the p53 coding sequence
Marcin
Task 1:
- Prepare PCR reaction to amplified p53 coding sequence.
Methods:
- DNA matrix dilution:
1 &mi;l of plasmid solution (isolated in 11.07.09) was taken and diluted with 99 mi&;l of MQ water
- PCR mixture composition:
- proper mixture 1: 0.25 μl primer 1 (50 nM; Oligo.pl), 0.25 μl primer 2 (50 nM; Oligo.pl), 1.5 μl dNTPs (20 μM ;Fermentas), 0.5 μl Pfu turbo polymerase (KNGiE), 2.5 μl Pfu Turbo Buffer (Fermentas), 2.5 μl MgSO4 (20 μM; Fermentas), 1 μl DNA matrix, 16.5 μl MQ water
- proprer mixture 2: 0.25 μl primer 1 (50 nM; Oligo.pl), 0.25 μl primer 2 (50 nM; Oligo.pl), 1.5 μl dNTPs (20 μM ;Fermentas), 0.5 μl Pfu turbo polymerase (KNGiE), 2.5 μl Pfu Turbo Buffer (Fermentas), 2.5 μl MgSO4 (20 μM; Fermentas), 2 μl DNA matrix, 15.5 μl MQ water
- Negative control: the same as proper mixture 1, the only distinction is lack of the DNA matrix.
- Program:
p53 (detailed destription is here)
Results:
- Clean-up the PCR products
Procedure: DNA was purified using the A&A clean-up kit. Detailed procedure is described here
Task 2:
- Restriction digest of p53 coding sequence.
Methods:
- Control digest using PvuII
- Reaction mixture composition: 1 μl purified PCR product, 0.5 μl PvuII (Fermentas), 2 μl Buffer Green (Fermentas), 15.5 μl MQ water
- Digest for subsequent cloning using XbaI
- Reaction mixture composition: 10 μl purified PCR product, 1 μl XbaI (Fermentas), 5 μl Buffer Green (Fermentas), 34.5 μl MQ water
- Both reaction were perform in the same condition:
Program:
digest:
1. 37°C - 3 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
Task 3:
- Cloning p53 coding sequence to pKS plasmid
Methods:
- Ligation mixture composition: 14 μl digested p53, 1.5 μl digested pKS, 5 μl ligation buffer (Invitrogen), 1 μl ligase T4
- Duration of ligation was about 12 hours
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