Team:Stockholm/20 August 2010

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(→Overnight culture of 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.
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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==Nina==
 
===Overnight culture of fusion protein===
===Overnight culture of fusion protein===

Revision as of 15:28, 25 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

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
  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

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

Bild13.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.

Digestion of protein A

I wanted to insert protein A in the peX vector in order to study its overexpression in BL21. I used the sample of protein A that had previously been digested with the restriction enzyme AgeI.

Digestion:

  • H2O 15 ul
  • 10 X fastdigest buffer 2 ul
  • DNA 2 ul
  • Restriction enzyme XbaI (fastdigest) 1 ul

I incubated in 37 °C for 5 min and then inactivated the restriction enzyme XbaI in 65 °C for 20 min.

Digestion of peX vector

I digested previously mini prepped peX vector in order for it to be able to ligate with the protein A gene that has been cut with the restriction enzymes AgeI and XbaI.

Digestion:

  • H2O 14 ul
  • 10 X fastdigest buffer 2 ul
  • DNA 2 ul
  • Restriction enzyme XbaI (fastdigest) 1 ul
  • Restriction enzyme AgeI (fastdigest) 1 ul

I incubated in 37 °C for 5 min and then inactivated the restriction enzyme XbaI in 65 °C for 20 min.

Agarose gel on digest products

I ran the digested products protein A and peX vector on an agarose gel 1 % 100 V in order to perform a gel clean up if the results would be good. This step would give me samples without any interupting bufferts from the digesion when perfoming a ligation in a later stage.

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

Arragement on gel:

Bild14.jpg

Bild15.jpg

Unfortunately I did not obtain satisfactory results on the digested protein A since I did not see any band representing the cut gene (180 nt)on the gel. In addition I believe the band representing the digested peX is unclear in its meaning. This is because I do not observe any small band that have been cut out of the digested vector. However, the band of the digested peX vector appears to correspond to the correct size after a digestion with AgeI and XbaI. It might just be that the cut site that should appear on the gel is in such a low concentration that it is not visiable on the gel, meaning still that the vector has become digested properly.

I continued with a gel clean up of only the digested peX vector and I re-did a digestion of the gene protein A.

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