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Team:Warsaw/Calendar-Main/21 July 2009
From 2009.igem.org
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Cloning of hly gene into pKSII+ vector
Kama
- chemocompetent E. coli dH5α were incubated on ice for 15 minutes
- 15μl of ligation mixture was added (and 15μl of control ligation, without insert)
- 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 800μl 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 (diluted 10* and without dilution)
Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2
Ania
Tasks:
- Alkaline lysis of the culture containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid.
- Digest of [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid with XbaI/PstI to obtain the insert
- Digest of [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_R0051 - cI regulated Promoter] on the pSB1A2 ampicillin resistant plasmid with SpeI/PstI to obtain the insert
- Gel
- DNA samples were cut out and frozen in the seperate eppendorfs (still within the agarose gel).
[image:
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Results:
[image:
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Comments:
- There is a vector with PcI clearly visible in the first two samples. The PcI is too short to be visible on its own. The insert, that is RBS.3+LacI is visible as the 1200 band in the last three samples. The vector from two first and the insert from two last samples were cut out of the gel.
Cloning of the mgtc promoter into the pKSII+ plasmid
Kamil
Tasks:
- Ligation verification
- Bacteria transformation
Methods:
- Correctness of the ligation was verified with gel electrophoresis on 1% agarose gel.
A 200μl batch of chemocompetent bacteria was transformed with 10μl of ligation mix and incubated overnight on petri dishes containing LB medium supplemented with ampicilin, X-gal and IPTG. (see protocols section for details on transformation).
Cloning of 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:
Composition of the mixtures was not changed. Detailed desciption is here (in the section "Cloning of 53 coding sequence").
- Program:
p53 (detailed destription is here)
Results:
No product obtained
Comment:
It is most likely that Pfu polimerase has been degradated. I decide to prepare this PCR reaction using the Phusion polimerase (NEB) which was kindly provided by dr. Dziembowski.
- 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) 0.25 μl Phusion polymerase (NEB) 5 μl Phusion Buffer (NEB) 2.5 μl MgSO4 (20 μM; Fermentas) 1 μl DNA template 14.5 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) 0.25 μl Phusion polymerase (NEB) 5 μl Phusion Buffer (NEB) 2.5 μl MgSO4 (20 μM; Fermentas) 2 μl DNA template 13.5 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:
PCR product from mixture 2.
Lack of the product from mixture 1
due to high concentration of the template.
Lack of the product from mixture 1
due to high concentration of the template.
Task 2:
- Clean-up the PCR product and subsequent digest
Methods:
- DNA was purified using the A&A clean-up kit. Detailed procedure is described here
- 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
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
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