Team:Stockholm/20 August 2010

From 2010.igem.org

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(Transformation)
(Agarose gel on fusion protein)
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Lane nr 3 looks like it could represent the wanted band of the fusion protein by protein A and IgG protease, which could be about 1300 nt.
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===Overnight culture of fusion protein===
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I inoculated colony nr 3 of the dish with the fusion protein into 12 ml LB and 24 ul chloramphenicol (50 mg/ml). This was incubated in 37 °C in shake overnight.

Revision as of 11:35, 25 August 2010


Contents


Hassan

midnight update, after a fast recovery from sickness!

Version 0.5.1


Version 0.6.1


Mimmi

pMA

Primer dilution

pMA_VF 91µl sH2O --> 100µM 10µl + 90µl sH2O --> 10µM
pMA_VR 91µl sH2O --> 100µM 10µl + 90µl sH2O --> 10µM


pMA/pEX

verification PCR

pEX pMA
Mix (µl) X6 X3 primers conditions
sH2O 22.5 135 67.5 pEX_VF time °C
F primer 1 6 3 pEX_VR 2m 94
R primer 1 6 3 pMA_VF 30s 94 )
DNA 0.5 5X0.5 2X0.5 pMA_VR 30s 60 > 30 cycles
tot 25µl 250µl 75µl 2m30s 72 )
10m 72
oo 10

Andreas

Transformation results

From 19/8 transformations

  • pEX.RFP: Good yield of red (positive) and white (negative) colonies.
  • pSB1K3.SOD⋅His: Very few colonies
  • pSB1K3.yCCS.His: Very few colonies
    • Km probably not feasible with quick transformation.

Colony PCR of pEX.RFP, pEX and pMA.His

Picked four pEX.RFP colonies for PCR verification. Also ran PCR on the pEX and pMA.His clones from 19/8. PCR and gel run by Mimmi (see above).

Results
  • pMA.His clone verified for correct insert.
  • pEX and pEX.RFP need to be re-run, as they did not result in any bands.

ON cultures

  • pMA.His
  • pEX (not yet verified)
  • pEX.RFP 3 (not yet verified)

3 ml LB + 100 Amp. 30 °C ON.

Plasmid prep

From 19/8 ON cultures

  • E.Z.N.A. Plasmid Mini Prep kit.
  • 70 μl elution volume
DNA concentrations
Sample Conc. [ng/μl] A260/A280
pSB1K3.BBa_J04450 103.8 1.94
pEX 55.52 1.89
pMA.His 85.67 1.87

Assembly of His⋅SOD/yCCS into pSB1K3

Step I of C-CPP cloning strategy (19/8)

Digestions

[pSB1C3.m-yCCS 1] = 101.2 ng/μl
[pSB1C3.m-SOD 2] = 105.5 ng/μl

[μl] pMA.His pSB1C3.m-yCCS 1 pSB1C3.m-SOD 1 Inc.: 37 °C,
0:30 (FD)
or
1:30 (NgoMIV)
10X FD buffer 3 3 3
dH2O 6.7† 6 7
2 μg DNA 23.3 20 19
FD EcoRI 0.5
FD PstI 1 1
FD AgeI 0.5
NgoMIV 1 1
  3030 30

Miscalculation

Ligations

Without prior enzyme inactivation or DNA purification.

[μl] Lig pSB1K3.
His⋅SOD
Lig pSB1K3.
His⋅yCCS
Vector/insert ratio:
1:3.

Inc.: 22 °C, 0:10
100 ng vector 2.2 2.2
His insert 4.0 4.0
SOD insert 5.1
yCCS 5.7
5X Rapid Lig. buf. 4 4
dH2O 2.7 2.1
T4 DNA ligase 1 1
  20 20

Enzyme inactivation (not PstI): 80 °C, 20 min.

Transformation

Standard transformation protocol.

  • 1 μl ligation mix
    • Lig pSB1K3.His⋅SOD
    • Lig pSB1K3.His⋅yCCS
  • Cells plated onto 50 Km LB agar plates

Nina

Colony PCR on fusion protein

I performed a colony PCR on the dish with fusion proteins.

PCR mix for 5 tubes:

  • MgCl 5 ul
  • Buffer 5X 50 ul
  • dNTP 5 ul
  • primer revers for IgG protease 15 ul
  • primer VF2 15 ul
  • polymerase PjuX7 5 ul
  • H2O 150 ul

PCR prgm:

Agarose gel on fusion protein

I ran the PCR product on an 1 % agarose gel 100 V in order to verify that I have bands corresponding to a correct size of the fusion protein.

Ladder: GeneRuler™ DNA Ladder Mix, ready-to-use, 100-10,000 bp Fermentas

Arrangement on gel:

Bild11.jpg

Bild12.jpg

Lane nr 3 looks like it could represent the wanted band of the fusion protein by protein A and IgG protease, which could be about 1300 nt.

Overnight culture of fusion protein

I inoculated colony nr 3 of the dish with the fusion protein into 12 ml LB and 24 ul chloramphenicol (50 mg/ml). This was incubated in 37 °C in shake overnight.