Team:Stockholm/30 September 2010

From 2010.igem.org

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[[Image:Ss1.jpg]]
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== Mimmi ==
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----
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=== Expression ===
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*yCCS expression looks very fine!
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*no SOD
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**much to low concentration loaded?
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**try E-colves for more air
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**taking samples after both 0.5h and 3h if the protein becomes degraded after time
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----
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=== SOD operon ===
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==== Plasmid prep. and sequencing ====
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*Follow E.T.Z.N.A plasmid prep. protocol
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**wash 1x wash buffer
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**eluate in 50µl
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*Measure DNA concentration
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pSB1K3.nLMWP.SOD.his.RBS.yCCS 2
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pSB1K3.nLMWP.SOD.his.RBS.yCCS 3
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pSB1K3.nTAT.SOD.his.RBS.yCCS 2
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pSB1K3.nTAT.SOD.his.RBS.yCCS 3
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pSB1K3.nTra10.SOD.his.RBS.yCCS 1
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pSB1K3.nTra10.SOD.his.RBS.yCCS 2
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*Send for sequencing...
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=== Over expression ===
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*Start ON culture
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**SOD
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**SOD.his
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**his.SOD
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**nTAT.SOD.his

Revision as of 23:04, 25 October 2010

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↳   Notebook Safety Protocols


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

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 inoculated 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

Laddermixmassruler.jpg

Arragement on gel:

Ss.jpg

Gel clean up

I performed a gel clean up according to the procedure described in protocols.

The measurements of the cut gel bands, addition of kit solutions and incubation time:

  • All bands had a weight of approximately 150 mg. 150 mg * 3 = 450 ul QXI
  • I added 30 ul of QIAEXII to all samples
  • All samples were incubated for 5 minutes at RT

Concentration measurments

I measured with a spectrophotometer the concentration of the gel clean up samples.

Ss1.jpg






Mimmi

Expression

  • yCCS expression looks very fine!
  • no SOD
    • much to low concentration loaded?
    • try E-colves for more air
    • taking samples after both 0.5h and 3h if the protein becomes degraded after time


SOD operon

Plasmid prep. and sequencing

  • Follow E.T.Z.N.A plasmid prep. protocol
    • wash 1x wash buffer
    • eluate in 50µl
  • Measure DNA concentration


pSB1K3.nLMWP.SOD.his.RBS.yCCS 2

pSB1K3.nLMWP.SOD.his.RBS.yCCS 3

pSB1K3.nTAT.SOD.his.RBS.yCCS 2

pSB1K3.nTAT.SOD.his.RBS.yCCS 3

pSB1K3.nTra10.SOD.his.RBS.yCCS 1

pSB1K3.nTra10.SOD.his.RBS.yCCS 2


  • Send for sequencing...


Over expression

  • Start ON culture
    • SOD
    • SOD.his
    • his.SOD
    • nTAT.SOD.his
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