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

Creating devices to test promoters in E. coli strains (devices [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025])

Franek

Tasks:

• Digestion of [http://partsregistry.org/Part:BBa_R0010 BBa_R0010 - lacI regulated promoter], [http://partsregistry.org/Part:BBa_R0080 BBa_R0080 - AraC regulated promoter] and [http://partsregistry.org/Part:BBa_ E0840 E0840 – RBS + GFP + Rerminator] bricks

• Ligation of [http://partsregistry.org/Part:BBa_R0010 lacI regulated promoter] and [http://partsregistry.org/Part:BBa_R0080 AraC regulated promoter] with [http://partsregistry.org/Part:BBa_ E0840 E0840]

Methods:

• DNA containing [http://partsregistry.org/Part:BBa_R0080 pAraC] and [http://partsregistry.org/Part:BBa_R0010 placI] was digested with SpeI and PstI enzymes. Digestion mix contained 10µl of extracted DNA, 2µl of Fermentas Tango buffer, 0.5µl of each enzyme and water added to obtain 20µl total volume. Both mixes were incubated for 3h at 37°C.

• DNA containing [http://partsregistry.org/Part:BBa_ E0840 E0840] was digested with XbaI and PstI enzymes. Digestion mix contained 26µl of extracted DNA, 3µl of Fermentas Tango buffer, 0.5µl of each enzyme. Two identical mixes created this way were incubated for 3h at 37°C.

• Enzymes in all 4 mixes were inactivated through incubation at 80°C for 20 min.

• Two ligation mixes were prepared. Each contained 30µl of [http://partsregistry.org/Part:BBa_R0080 pAraC]/ [http://partsregistry.org/Part:BBa_R0010 placI] digestion mix, 20µl of [http://partsregistry.org/Part:BBa_ E0840 E0840] mix, 5µl of dNTPs and 2µl of ligase. Both were left at 16°C for over night incubation.

Results:

• Positive selection will be made according to fluorescence in UV light