Team:Stockholm/30 September 2010

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(Difference between revisions)
(→Andreas)
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**pEX.nTAT⋅SOD⋅His (Top10; Amp 100)
**pEX.nTAT⋅SOD⋅His (Top10; Amp 100)
**pSB1K3.BBa_J04450 (Top10; Km 50)
**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
 +
 +
[[Image:laddermixmassruler.jpg|200px]]
 +
 +
Arrangement on gel:
 +
 +
[[Image:B1.jpg]]
 +
 +
===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.

Revision as of 07:02, 7 October 2010

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Contents

Andreas

Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX

Digestions

  1. pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS
    • Clones 2 & 3
  2. pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS
    • Clones 1 & 2
  3. 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

Laddermixmassruler.jpg

Arrangement on gel:

B1.jpg

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.

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