Team:Stockholm/4 October 2010

From 2010.igem.org

(Difference between revisions)
(→Andreas)
(→Transformations)
Line 121: Line 121:
**1 μl
**1 μl
**Cm 25
**Cm 25
 +
 +
----
 +
 +
----
 +
==Nina==
 +
 +
===Mini prep on IgG_Tra10_N#6===
 +
 +
I performed a mini prep on yesterday's inoculated IgG_Tra10_N colony # 6 in 12 ml LB with 24 ul chloramphenicol. The procedure was according to the method described in protocols.
 +
 +
===Overday culture of SOD===
 +
 +
I put an overday culture of SOD-peX, His-SOD, SOD-His, SOD_TAT_Ntermin and as a positive control yCC-peX from Andrea's glycerol stocks in 12 ml LB with 24 ul amphicillin. I inoculated with a big pipett tip amount in order to have the culture reaching OD 0.6 during the day of laboration.
 +
 +
The reason for why I did this culture was because I had talked to Mimmi about her overexpressions about these constructs and she hadn't got any satisfying results on her gels beside from the yCC sample (which became my positive control). I wanted therefore to check if I would also obtain her type of outcome when overexpressing the proteins of interest.
 +
 +
===Gene digestions===
 +
 +
I performed digestions of my genes of interest in order to do a gel clean up and follow with a ligation that should yield many positive cloning results.
 +
 +
Digestions:
 +
 +
*Fusion_EA_His # 1 & 3 10 ul
 +
*Fast digestion buffer 10 X 2 ul
 +
*H2O 6 ul
 +
*Restriction enzyme NgoMVI 1 ul
 +
*Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)
 +
 +
 +
*Fusion_NS_His # 2 10 ul
 +
*Fast digestion buffer 10 X 2 ul
 +
*H2O 6 ul
 +
*Restriction enzyme AgeI 1 ul
 +
*Restriction enzyme EcoRI 1 ul (Added after 1.5 h in 37 °C)
 +
 +
 +
*Protein A_EA_His # 5 10 ul
 +
*Fast digestion buffer 10 X 2 ul
 +
*H2O 6 ul
 +
*Restriction enzyme NgoMVI 1 ul
 +
*Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)
 +
 +
 +
*IgG protease_EA_His # 5 10 ul
 +
*Fast digestion buffer 10 X 2 ul
 +
*H2O 6 ul
 +
*Restriction enzyme NgoMVI 1 ul
 +
*Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)
 +
 +
 +
*IgG protease_Tra10_Ntermin # 4 & 6 10 ul
 +
*Fast digestion buffer 10 X 2 ul
 +
*H2O 6 ul
 +
*Restriction enzyme XbaI 1 ul
 +
*Restriction enzyme PstI 1 ul
 +
 +
Incubate in 37 °C for 30 minutes.
 +
 +
===Agarose gel on digests===
 +
 +
I ran an agarose gel 1 % 100 V on the digested samples in order to check if they have been digested by the enzymes and followed by cutting the bands of interest out of the gel with a scalpel over a UV-lamp for a gel clean up.
 +
 +
Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp
 +
 +
[[Image:laddermixmassruler.jpg|200px]]
 +
 +
Arrangement on gels:
 +
 +
[[Image:Az.jpg]]
 +
 +
===Gel clean up===
 +
 +
I performed a gel clean up of the digested genes. All except IgGEA#5 were digested and cut out of the gel. I performed the clean up according to the method described in protocols.
 +
 +
The measurements of the cut gel bands, addition of kit solutions and incubation time:
 +
 +
*All bands had a weight of aproximately 0.12 g (120 mg). 120 mg * 3 = 360 ul QXI
 +
 +
*I added 10 ul of QIAEXII to all samples
 +
 +
*All samples were incubated for 5 minutes at RT
 +
 +
*I performed step 11 in the procedure description
 +
 +
===Concentration measurments===
 +
 +
I measured with a spectrophotometer the concentration of the gel clean up samples.
 +
 +
[[Image:Nm.jpg]]
 +
 +
===Sending for sequencing===
 +
 +
I sent IgG_Tra10_Ntermin#6 for sequencing. I mixed 15 ul of the miniprep sample and 1.5 ul of Forward bank vector verification primer (VF).
 +
 +
* ASB0045 898

Revision as of 06:05, 7 October 2010

Home Project Idea Lab Work Results Modelling Team       Follow us on and        2010.igem.org

↳   Notebook Safety Protocols


Contents

Andreas

Transfer of pEX.nCPP⋅SOD⋅His to BL21

Gel verification

Colony PCR gel verification of BL21 clones carrying pEX.nLMWP⋅SOD⋅His (1) and pEX.nTra10⋅SOD⋅His (2) plasmids.
4 μl λ; 5 μl sample;
λ = O'GeneRuler 1 kb DNA ladder.

Re-run of 2/10 BL21 samples

  1. BL21 pEX.nLMWP⋅SOD⋅His: A & B
  2. BL21 pEX.nTra10⋅SOD⋅His: A & B

1 % agarose, 120 V

Expected bands:

  1. 744 bp
  2. 765 bp

Results

  1. Both clones verified
  2. Clone A verified; weak band for clone B

ON cultures

  • 3 ml LB, 30 °C
    1. A (BL21 pEX.nLMWP⋅SOD⋅His)
    2. A (BL21 pEX.nTra10⋅SOD⋅His)

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

Colony PCR

Picked 2 new colonies of each of the two constructs transformed 30/8:

  • 5. pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
  • 6. pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B

Standard colony PCR settings

  • Elongation time: 2:00

Gel verification

Colony PCR gel verification of pEX.nCPP⋅SOD⋅His.RBS.yCCS operon clones.
4 μl λ; 5 μl sample;
λ = O'GeneRuler 1 kb DNA ladder.

0.8 % agarose, 100 V

Expected bands:

  • 5. 1553 bp
  • 6. 1553 bp

Results

  • 5. Relevant band for clone B; too large insert (double?) for clone A.
  • 6. Relevant band for clone B; too large insert (double?) for clone A.

ON cultures

  • 5 ml LB, 37 °C, 250 rpm
    • pEX.nTAT⋅SOD⋅His.RBS.yCCS 2: A
    • pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: B
    • pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: B
    • pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: B
    • pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: B
    • pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: B

Verification of pSB1x3 plasmids

Due to some strange growth results with our stock plasmids (pSB1x3.BBa_J04450), I decided to verify their antibiotic resistance. Restreaked clones of the following plasmids (w/ BBa_J04450 inserts) onto Amp 100, Km 50 and Cm 25 plates:

  • pSB1A3
  • pSB1C3
  • pSB1K3
  • pSB1AC3
  • pSB1AK3

Assembly of His⋅SOD⋅cCPP constructs

Continued from 1/10

Received cCPPs (cTra10, cTAT and cLMWP) in pSB1C3 plasmids, digested with EcoRI and NgoMIV, from Johan.

Ligations

  • Vectors:
    1. Dig pSB1C3.cTra10 E+N
    2. Dig pSB1C3.cTAT E+N
    3. Dig pSB1C3.cLMWP E+N
  • Insert: Dig pMA.His⋅SOD E+A
  1 2 3
10X T4 Ligase buffer 2 2 2
Vector DNA 1 1 1
Insert DNA 5 5 5
dH2O 11 11 11
T4 DNA ligase 1 1 1
  20 μl 20 μl 20 μl
  • Incubation: 22 °C, 15 min

Transformations

  1. pSB1C3.His⋅SOD⋅cTra10
  2. pSB1C3.His⋅SOD⋅cTAT
  3. pSB1C3.His⋅SOD⋅cLMWP
  • Standard transformation
    • 1 μl
    • Cm 25

Nina

Mini prep on IgG_Tra10_N#6

I performed a mini prep on yesterday's inoculated IgG_Tra10_N colony # 6 in 12 ml LB with 24 ul chloramphenicol. The procedure was according to the method described in protocols.

Overday culture of SOD

I put an overday culture of SOD-peX, His-SOD, SOD-His, SOD_TAT_Ntermin and as a positive control yCC-peX from Andrea's glycerol stocks in 12 ml LB with 24 ul amphicillin. I inoculated with a big pipett tip amount in order to have the culture reaching OD 0.6 during the day of laboration.

The reason for why I did this culture was because I had talked to Mimmi about her overexpressions about these constructs and she hadn't got any satisfying results on her gels beside from the yCC sample (which became my positive control). I wanted therefore to check if I would also obtain her type of outcome when overexpressing the proteins of interest.

Gene digestions

I performed digestions of my genes of interest in order to do a gel clean up and follow with a ligation that should yield many positive cloning results.

Digestions:

  • Fusion_EA_His # 1 & 3 10 ul
  • Fast digestion buffer 10 X 2 ul
  • H2O 6 ul
  • Restriction enzyme NgoMVI 1 ul
  • Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)


  • Fusion_NS_His # 2 10 ul
  • Fast digestion buffer 10 X 2 ul
  • H2O 6 ul
  • Restriction enzyme AgeI 1 ul
  • Restriction enzyme EcoRI 1 ul (Added after 1.5 h in 37 °C)


  • Protein A_EA_His # 5 10 ul
  • Fast digestion buffer 10 X 2 ul
  • H2O 6 ul
  • Restriction enzyme NgoMVI 1 ul
  • Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)


  • IgG protease_EA_His # 5 10 ul
  • Fast digestion buffer 10 X 2 ul
  • H2O 6 ul
  • Restriction enzyme NgoMVI 1 ul
  • Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)


  • IgG protease_Tra10_Ntermin # 4 & 6 10 ul
  • Fast digestion buffer 10 X 2 ul
  • H2O 6 ul
  • Restriction enzyme XbaI 1 ul
  • Restriction enzyme PstI 1 ul

Incubate in 37 °C for 30 minutes.

Agarose gel on digests

I ran an agarose gel 1 % 100 V on the digested samples in order to check if they have been digested by the enzymes and followed by cutting the bands of interest out of the gel with a scalpel over a UV-lamp for a gel clean up.

Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp

Laddermixmassruler.jpg

Arrangement on gels:

Az.jpg

Gel clean up

I performed a gel clean up of the digested genes. All except IgGEA#5 were digested and cut out of the gel. I performed the clean up according to the method described in protocols.

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

  • All bands had a weight of aproximately 0.12 g (120 mg). 120 mg * 3 = 360 ul QXI
  • I added 10 ul of QIAEXII to all samples
  • All samples were incubated for 5 minutes at RT
  • I performed step 11 in the procedure description

Concentration measurments

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

Nm.jpg

Sending for sequencing

I sent IgG_Tra10_Ntermin#6 for sequencing. I mixed 15 ul of the miniprep sample and 1.5 ul of Forward bank vector verification primer (VF).

  • ASB0045 898
Retrieved from "http://2010.igem.org/Team:Stockholm/4_October_2010"