Team:Stockholm/20 August 2010

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(Difference between revisions)
(→Andreas)
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!Lig pSB1K3.<br />His&sdot;SOD
!Lig pSB1K3.<br />His&sdot;SOD
!Lig pSB1K3.<br />His&sdot;yCCS
!Lig pSB1K3.<br />His&sdot;yCCS
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|rowspan="9" align="center"|Vector/insert ratio:<br />1:3.<br /><br /> Inc.: 22 &deg;C, 0:10
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|rowspan="9" align="center" width="100"|Vector/insert ratio:<br />1:3.<br /><br /> Inc.: 22 &deg;C, 0:10
|-
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|'''100 ng vector'''
|'''100 ng vector'''

Revision as of 22:04, 21 August 2010

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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

Assembly of CPP⋅protein⋅His constructs

Continued from 19/8

Transformation results

  • 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.

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
  30† 30 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

ON cultures

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

3 ml LB + 100 Amp. 30 °C ON.

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