"
Team:Stockholm/30 September 2010
From 2010.igem.org
NinaSchiller (Talk | contribs) (→Gel clean up) |
NinaSchiller (Talk | contribs) (→Overnight culture) |
||
| Line 226: | Line 226: | ||
I ioculated IgG protease_Tra10 # 4 & 6 in each 12 ml LB falcon tubes with 24 ul chloramphenicol. | I ioculated IgG protease_Tra10 # 4 & 6 in each 12 ml LB falcon tubes with 24 ul chloramphenicol. | ||
| + | |||
| + | ===Digestion of vectors with CPP=== | ||
| + | |||
| + | I digested bank vectors holding LMWP_N, TAT_N, Tra10_N, CPP1_C, TAT_C and CPP3_C to become followed by a gel clean up. | ||
| + | |||
| + | Digestion: | ||
| + | |||
| + | *DNA 20 ul | ||
| + | *Fast digest buffer 10X 2.2 ul | ||
| + | |||
| + | CPPs-Ntermin: | ||
| + | |||
| + | *Restriction enzyme AgeI 1 ul | ||
| + | *Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C) | ||
| + | |||
| + | CPPs-Ctermin: | ||
| + | |||
| + | *Restriction enzyme NgoMIV 1 ul | ||
| + | *Restriction enzyme EcoRI 1 ul (Added after 1.5 h in 37 °C) | ||
| + | |||
| + | ===Agarose gel on digests=== | ||
| + | |||
| + | I ran the digests on an agarose 1 % gel 80 V. | ||
| + | |||
| + | Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp | ||
| + | |||
| + | [[Image:laddermixmassruler.jpg|200px]] | ||
| + | |||
| + | Arragement on gel: | ||
| + | |||
| + | [[Image:Ss.jpg]] | ||
| + | |||
| + | ===Gel clean up=== | ||
| + | |||
| + | I performed a gel clean up according to the procedure described in protocols. | ||
Revision as of 07:48, 7 October 2010
| Home | Project Idea | Lab Work | Results | Modelling | Team | Follow us on  and 
| 2010.igem.org 
|
Contents |
Andreas
Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX
Digestions
- pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS
- Clones 2 & 3
- pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS
- Clones 1 & 2
- pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS
- Clones 2 & 3
| 1:2 | 1:3 | 2:1 | 2:2 | 3:2 | 3:3 | |
| 10X FastDigest buffer | 2 | 2 | 2 | 2 | 2 | 2 |
| DNA (1 μg) | 5.2 | 4.1 | 2.5 | 3.3 | 4 | 4.1 |
| dH2O | 10.8 | 11.9 | 13.5 | 12.7 | 12 | 11.9 |
| FD XbaI | 1 | 1 | 1 | 1 | 1 | 1 |
| FD PstI | 1 | 1 | 1 | 1 | 1 | 1 |
| 20 μl | 20 μl | 20 μl | 20 μl | 20 μl | 20 μl |
|---|
- Incubation: 37 °C, 1.45
- Inactivation: 80 °C, 20 min
Ligations
- Vector: [Dig pEX.RFP X+P
| pEX.1:2 | pEX.1:3 | pEX.2:1 | pEX.2:2 | pEX.3:2 | pEX.3:3 | |
| 10X T4 Ligase buffer | 2 | 2 | 2 | 2 | 2 | 2 |
| Vector DNA | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
| Insert DNA | 8 | 8 | 8 | 8 | 8 | 8 |
| dH2O | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 |
| T4 DNA ligase | 1 | 1 | 1 | 1 | 1 | 1 |
| 20 μl | 20 μl | 20 μl | 20 μl | 20 μl | 20 μl |
|---|
- Incubation: 22 °C, 15 min
Transformation
Quick transformation
- 1 μl ligation mix
- 50 μl 0.1 M IPTG
- pEX.1:2 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2)
- pEX.1:3 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2)
- pEX.2:1 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 1)
- pEX.2:2 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 2)
- pEX.3:2 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2)
- pEX.3:3 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3)
Transformation of BL21
Quick transformation
- 50 μl competent cells
- 0.5 μl plasmid
- pEX.nTra10⋅SOD⋅His
- pEX.nLMWP⋅SOD⋅His
ON cultures
- 3 ml LB + appropriate antibiotic; 30 °C
- pEX.nTAT⋅SOD⋅His (Top10; Amp 100)
- pSB1K3.BBa_J04450 (Top10; Km 50)
Nina
Send for sequencing
I sent samples for sequencing and the mixtures were 15 ul sample and 1.5 ul forward bank vector verification primer.
- Protein A in LMWP_Ntermin ASB0045 680
- Protein A in TAT_Ntermin ASB0045 679
- Protein A in Tra10_Ntermin ASB0045 678
Digestion of protein A and peX vector
I got a mini prep of the peX vector from Andreas with a concentration of 55.52 ng/ul. I cut this vector and protein A in CPPs_N vectors.
peX:
- Fast digest buffer 10X 3.4 ul
- DNA 30 ul
- Restriction enzyme XbaI 2 ul
- Restriction enzyme PstI 2 ul
Incubated in 37 °C for 30 min.
Protein A:
- Fast digest buffer 10X 2.2 ul
- DNA 20 ul
- Restriction enzyme XbaI 1 ul
- Restriction enzyme PstI 1 ul
Incubated in 37 °C for 30 min.
Agarose gel on digests
I ran the digested products on an agarose gel 1 % 100 V.
Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp
Arrangement on gel:
Gel clean up
I performed a gel clean up according to the procedures described in protocols.
On the gel it looked like it was only the peX vector that had been cut correctly and thus the protein A didn't seem to have been previously inserted correctly into the vectors holding the CPPs on the N-terminal. Therefore I only cut out and gel clean the peX vector for later use in overexpressions of our proteins.
The measurements of the cut gel bands, addition of kit solutions and incubation time:
- All bands had a weight of approximately 300 mg. 300 mg * 3 = 900 ul QXI
- I added 30 ul of QIAEXII to all samples
- All samples were incubated for 5 minutes at 50 °C
Miniprep
I performed a mini prep on Fusion EA # 1 and 3. Fusion AS I have to put a new overnight culture of since I accidentally dropped the solution and the material is now lost.
The procedure was according to the method described in protocols.
Overnight culture
I ioculated IgG protease_Tra10 # 4 & 6 in each 12 ml LB falcon tubes with 24 ul chloramphenicol.
Digestion of vectors with CPP
I digested bank vectors holding LMWP_N, TAT_N, Tra10_N, CPP1_C, TAT_C and CPP3_C to become followed by a gel clean up.
Digestion:
- DNA 20 ul
- Fast digest buffer 10X 2.2 ul
CPPs-Ntermin:
- Restriction enzyme AgeI 1 ul
- Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)
CPPs-Ctermin:
- Restriction enzyme NgoMIV 1 ul
- Restriction enzyme EcoRI 1 ul (Added after 1.5 h in 37 °C)
Agarose gel on digests
I ran the digests on an agarose 1 % gel 80 V.
Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp
Arragement on gel:
Gel clean up
I performed a gel clean up according to the procedure described in protocols.
