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Team:Warsaw/Calendar-Main/13 July 2009
From 2009.igem.org
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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 [http://www.aabiot.com/products/dna_purification/dna_fragments/clean_up/protocol_clean_up.pdf here]
After clean-up 1 μl of purified PCR product was loaded into gel and photographed. Unfortunatelly reaction 1 (2 μl of DNA matrix) was abortive - there is no product in the gel.
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 Tango (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 + Terminator]
- 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 under UV light
Cloning of the mgtc promoter into the pKSII+ plasmid
Kamil
Tasks:
- Plasmid assembly
Methods (the bulk technique):
- Plasmid digest mix was prepared as follows: 2ul Tango buffer (Fermentas), 3ul pKSII+ plasmid, 1ul XbaI enzyme, 1ul SmaI enzyme, the solution was topped up with H2O to the final volume of 20 ul.
- mgtc promoter digest mix was prepared as follows: 2ul Tango buffer (Fermentas), 10ul purified gene, 1ul XbaI enzyme, the solution was topped up with H2O to the final volume of 20 ul.
- The digest was kept for 3h at 37°C, and then the enzyme was inactivated for 15min. at 80°C.
The ligation mix was prepared as follows: both inactivated digests were mixed together and topped with 5ul of 30% PEG, 5ul 10mM ATP, 1,2ul Tango buffer (Fermentas) and 2ul of T4 ligase (Fermentas). The ligation was carried out in 18°C overnight (~18h) and then inactivated for 10min. at 65°C.
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