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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 template 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 template, 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 template, 15.5 μl MQ water
- Negative control: the same as proper mixture 1, the only distinction is lack of the DNA template.
- 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
- After clean-up 1 μl of purified PCR product was loaded into gel and photographed.
verification of PCR reaction and clean-up procedure
Comment:
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
control digest of p53 with PvuII
Restriction pattern confirms that sequence is correct
- 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:
- Cloning p53 coding sequence to pKS plasmid
- 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
- Digestion of BBa_R0010 - lacI regulated promoter, BBa_R0080 - AraC regulated promoter and E0840 – RBS + GFP + Terminator
- Ligation of lacI regulated promoter and AraC regulated promoter with E0840
- DNA containing pAraC and 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 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 pAraC/ placI digestion mix, 20µl of E0840 mix, 5µl of dNTPs and 2µl of ligase. Both were left at 16°C for over night incubation.
- Positive selection will be made according to fluorescence under UV light
- Plasmid assembly
- Plasmid digest mix was prepared as follows: 2μl Tango buffer (Fermentas), 3μl pKSII+ plasmid, 2μl XbaI enzyme, 2μl SmaI enzyme, the solution was topped up with H2O to the final volume of 20 ul.
- mgtc promoter digest mix was prepared as follows: 2μl Tango buffer (Fermentas), 10μl purified gene, 2μl 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 5μl of 30% PEG, 5μl 10mM ATP, 1,2μl Tango buffer (Fermentas) and 2μl of T4 ligase (Fermentas). The ligation was carried out in 18°C overnight (~18h) and then inactivated for 10min. at 65°C.
- chemocompetent E. coli dH5α were incubated on ice for 15 minutes
- ligation mixture was added
- bacteria were incubated with DNA on ice for 30 minutes
- heat shock was conducted (1 minute 42°C)
- bacteria were incubated on ice for 3 minutes
- After the heat shock 750μl of SOB medium was added
- Mixture with bacteria was incubated for 1 hour in 37°C
- 100μl of mixture was plated on medium containing ampicillin, X-Gal and IPTG
- The rest of mixture was rotated for 1 minute, pelet was
- Set up a liquid culture from traqnsformants containing BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid.
- Alkaline lysis of bacterial cultures to obtain plasmids containing following bricks:
- Digest of BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid (XbaI/PstI = prospective insert)
- Digest of BBa_B0032 - RBS.3 on the pSB1A2 ampicillin resistant plasmid (SpeI/PstI = prospective vector)
- Fail. Digestion mix was incubated overnight at room temperature, this might be the reason for the unproper digestion.
1 μl of the digest mixture was diluted to 10 μl and loaded into the gel.
PCR product after digest loaded into the gel
Task 3:
Methods:
Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025)
Franek
Tasks:
Methods:
Results:
Cloning of the mgtc promoter into the pKSII+ plasmid
Kamil
Tasks:
Methods (the bulk technique):
Transformation of E. coli with pKS/hly plasmid
Kama
Construction of K177012 operon1_part2
Ania
Tasks:
BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid.
Results:
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