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:

1. 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
2. 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
3. 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 [http://www.aabiot.com/products/dna_purification/dna_fragments/clean_up/protocol_clean_up.pdf 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 [http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf 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