Team:Stockholm/30 September 2010

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Revision as of 07:29, 7 October 2010

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.

@@ Line 211: / Line 211: @@
 The measurements of the cut gel bands, addition of kit solutions and incubation time:
-*All bands had a weight of aproximately 300 mg. 300 mg * 3 = 900 ul QXI
+*All bands had a weight of approximately 300 mg. 300 mg * 3 = 900 ul QXI
 *I added 30 ul of QIAEXII to all samples