Team:Warsaw/Calendar-Main/28 August 2009
From 2009.igem.org
Cloning of the mgtc promoter into the pSB1A3 plasmid
Kamil
Tasks:
- Amplification of the mgtc promoter using new primers
- pSB1A3 plasmid digest
Methods:
- The PCR mix was prepared as follows:
5μl buffer B (EURx)
2μl 5mM dNTPs (EURx)
5μl forward starter
5μl reverse starter
2μl OptiTaq polymerase (EURx)
2μl Salmonella matrix
29 μl H2O- PCR programme:
4min 95°C
(30s 95°C, 35s 48°C, 40s 72°C)x28
(30s 95°C, 35s 58°C, 40s 72°C)x28
10min 72°C
~ 4°C- The PCR results were visualised with gel electrophoresis on 1% agarose gel.
- The digest mix was prepared as follows:
60μl plasmid isolation
7μl Buffer O (Fermentas)
1μl EcoRI enzyme
1μl PstI enzyme
1μl H2O- The digest was carried out in 37°C for 3h and inactivated for 15 min. in 80°C.
- 1μl of CIAP enzyme was added after the inactivation and allowed to work for 1h in 37°C and was later inactivated for 15 min. in 85°C.
- The resulting mix was separated on 1% agarose gel alongside the PCR and then selected bands were extracted for purification.
Results:
- No gel picture, but the PCR worked so great that I decided not to irradiate the gel with UV light more than it was necessary for band extraction.
Conclusions:
- The temperatures were chosen... wisely.
Cloning of the cro-box into the pSB1A3 plasmidKamil
Tasks:
- Cro-box digest
Methods:
- The digest mix was prepared as follows:
20μl plasmid isolation
3μl Buffer O (Fermentas)
1μl EcoRI enzyme
1μl PstI enzyme
5μl H2O
Assembly of endosomal detection operonMarcin
Task 1:
- Gel-outs of following constructs digested on 17.08.09:
Methods:
- All gel-outs were performed using the EurX gel-out kit according to the manual
Results:
- Purification of insert assembled with C0040 and RBS failed because of very low DNA concentration after plasmid isolation
Comment:
Isolation of plasmid which contain C0051 has very low field so the entire amount of DNA digested from the plasmid was insufficient to perform gel-out. It is obligatory to digest this construct one more time
Task 2:
- Digestion of following construct:
20 μl purified plasmid DNA product 1 μl XbaI (Fermentas) 1 μl PstI (Fermentas) 2 μl Buffer Tango (Fermentas) 15 μl MQ water
- Digest was performed about six hours and subsequently thermal inactivated
Task 3:
- Gel-outs of construct described in Task 2
Methods:
- All gel-outs were performed using the EurX gel-out kit according to the manual
Results:
- Purification of insert assembled with C0040 and RBS failed because of very low DNA concentration after plasmid isolation
Comment:
Isolation of plasmid which contain C0051 has very low field so the entire amount of DNA digested from the plasmid may be insufficient for efficient gel-out. Although I decided to continue the task
Task 4:
Methods:
- Ligation mixtures composition:
30 μl digested insert 10 μl digested vector 5 μl Tango buffer(Fermentas) 3 μl dNTPs mixture (EurX, concentration 5 mM) 2 μl ligase T4 (Fermentas)
- Duration of ligation was about 18 hours; reaction was conducted in 19 °c (approximately).
- In the next step ligated samples were thermally inactivated via heating in 80 °c for 20 minutes
Making of the plac-RBS-llo-intA partJarek
Tasks:
- Digestion of pUC19-intA with PstI and CaiI endonucleases, and plac-RBS-llo with PstI (this sample was additionaly defosforylated during digestion).
- Separation of digested fragments in 0,8% agarose gel
- Another digestion of pUC19-intA and plac-RBS-llo with the same enzymes
Results:
- After seting electrophoresis I found out that the digestion was prepared wrong, also gel went wrong.
Ania
Tasks:
April M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 May M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 June M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 July M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 August M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 September M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 October M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
- PCR programme: