Team:Stockholm/28 September 2010

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{{Stockholm/Top2}}
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==Andreas==
==Andreas==
 +
===Assembly and cloning===
 +
====Gel verification====
 +
[[image:ColPCR_pEX.nCPP.SH_28sep.png‎|200px|thumb|right|'''Colony PCR gel verification of pEX.nCPP&sdot;SOD&sdot;His constructs.'''<br />3 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
 +
[[image:ColPCR_nCPP.SH.Ry_28sep.png‎|200px|thumb|right|'''Colony PCR gel verification of nCPP&sdot;SOD&sdot;His.RBS.yCCS constructs.'''<br />3 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
 +
 +
''Re-run of 27/9 colony PCRs.''
 +
 +
'''Gel 1'''<br />
 +
1 % agarose, 110 V
 +
 +
'''Gel 2'''<br />
 +
0.8 % agarose, 90 V
 +
 +
'''Expected bands'''
 +
#'''pEX.N-LMWP&sdot;SOD&sdot;His (K):''' 744 bp
 +
#'''pEX.N-TAT&sdot;SOD&sdot;His:''' 735 bp
 +
#'''pEX.N-Tra10&sdot;SOD&sdot;His:''' 765 bp
 +
#'''pEX.N-LMWP&sdot;SOD&sdot;His (C):''' 744 bp
 +
#'''pSB1K3.N-LMWP&sdot;SOD&sdot;His.RBS.yCCS:''' 1645 bp
 +
#'''pSB1K3.N-TAT&sdot;SOD&sdot;His.RBS.yCCS:''' 1636 bp
 +
#'''pSB1K3.N-Tra10&sdot;SOD&sdot;His.RBS.yCCS:''' 1666 bp
 +
#'''pSB1C3.N-LMWP&sdot;SOD&sdot;His.RBS.yCCS:''' 1645 bp
 +
#'''BL21 pEX.N-TAT&sdot;SOD&sdot;His 3:''' 735 bp
 +
#'''BL21 pEX.N-TAT&sdot;SOD&sdot;His 4:''' 735 bp
 +
 +
'''Results'''<br />
 +
Listing clones of seemingly correct sizes, thereby potentially correct constructs:
 +
#1
 +
#1 & 2
 +
#1 & 2
 +
#No correct clones
 +
#2, 3 & 4
 +
#1, 2 & 3
 +
#1, 2, 3 & 4
 +
#No correct clones
 +
#1 & 2
 +
#No correct clones
 +
 +
For further verification constructs should be sent for sequencing. However, we will await the sequencing results from samples sent 27/9, as they were also used for building these new assemblies.
 +
 +
====ON cultures====
 +
Set ON cultures for plasmid prep (constructs 1-8) and glycerol stock (9)
 +
*5 ml LB + appr. antibiotic (Amp 100 or Km 50); 37 &deg;C, 220 rpm
 +
:1. 1
 +
:2. 1
 +
:3. 1
 +
:4. -
 +
:5. 2 & 3
 +
:6. 2 & 3
 +
:7. 1 & 2
 +
:8. -
 +
*3 ml LB + Amp 100; 30 &deg;C
 +
:9. 1
 +
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== Gel for Andreas colony-PCR ===
 +
 +
*Make two gels, one 1% and one 0.8% agarose
 +
 +
*Load samples in number order for Andreas to analyze
 +
 +
 +
 +
 +
=== Over expression ===
 +
 +
*Start up cultures
 +
{| align="right"
 +
| '''DNA'''
 +
|-
 +
| pEX.SOD
 +
|-
 +
| pEX.yCCS
 +
|-
 +
| pEX.SOD.his
 +
|-
 +
| pEX.his.SOD
 +
|}
 +
**10ml LB<sub>AMP</sub> + 100µl old ON culture
 +
 +
*At OD=0.6 (3h) add 1mM IPTG
 +
**Take sample at 0h and 3h
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***1ml culture
 +
***spinn down cells
 +
***remove LB
 +
***add 100µl sample buffer
 +
***freeze
 +
***heat to 95&deg;C for 10min
 +
 +
==Nina==
 +
 +
===Colony PCR screen===
 +
 +
I performed a PCR screen on the colonies from yesterday's transformation.
 +
 +
pEX Master Mix 15 tubes:
 +
 +
*MgCl2 7.5 ul
 +
*phusion buffer 5X 75 ul
 +
*dNTP 7.5 ul
 +
*PrimerF 22.5 ul
 +
*PrimerR 22.5 ul
 +
*polymerase 7.5 ul
 +
*H2O 225 ul
 +
 +
Add 24 ul/PCR tube
 +
 +
pMa Master Mix 10 tubes:
 +
 +
*MgCl2 5 ul
 +
*phusion buffer 5X 50 ul
 +
*dNTP 5 ul
 +
*PrimerF 15 ul
 +
*PrimerR 15 ul
 +
*polymerase 5 ul
 +
*H2O 150 ul
 +
 +
Add 24 ul/PCR tube
 +
 +
Bankvector C Master Mix 25 tubes:
 +
 +
*MgCl2 12.5 ul
 +
*phusion buffer 5X 125 ul
 +
*dNTP 12.5 ul
 +
*PrimerF 37.5 ul
 +
*PrimerR 37.5 ul
 +
*polymerase 12.5 ul
 +
*H2O 375 ul
 +
 +
Add 24 ul/PCR tube
 +
 +
{{Stockholm/Footer}}

Latest revision as of 11:09, 26 October 2010

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Contents

Andreas

Assembly and cloning

Gel verification

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

Re-run of 27/9 colony PCRs.

Gel 1
1 % agarose, 110 V

Gel 2
0.8 % agarose, 90 V

Expected bands

  1. pEX.N-LMWP⋅SOD⋅His (K): 744 bp
  2. pEX.N-TAT⋅SOD⋅His: 735 bp
  3. pEX.N-Tra10⋅SOD⋅His: 765 bp
  4. pEX.N-LMWP⋅SOD⋅His (C): 744 bp
  5. pSB1K3.N-LMWP⋅SOD⋅His.RBS.yCCS: 1645 bp
  6. pSB1K3.N-TAT⋅SOD⋅His.RBS.yCCS: 1636 bp
  7. pSB1K3.N-Tra10⋅SOD⋅His.RBS.yCCS: 1666 bp
  8. pSB1C3.N-LMWP⋅SOD⋅His.RBS.yCCS: 1645 bp
  9. BL21 pEX.N-TAT⋅SOD⋅His 3: 735 bp
  10. BL21 pEX.N-TAT⋅SOD⋅His 4: 735 bp

Results
Listing clones of seemingly correct sizes, thereby potentially correct constructs:

  1. 1
  2. 1 & 2
  3. 1 & 2
  4. No correct clones
  5. 2, 3 & 4
  6. 1, 2 & 3
  7. 1, 2, 3 & 4
  8. No correct clones
  9. 1 & 2
  10. No correct clones

For further verification constructs should be sent for sequencing. However, we will await the sequencing results from samples sent 27/9, as they were also used for building these new assemblies.

ON cultures

Set ON cultures for plasmid prep (constructs 1-8) and glycerol stock (9)

  • 5 ml LB + appr. antibiotic (Amp 100 or Km 50); 37 °C, 220 rpm
1. 1
2. 1
3. 1
4. -
5. 2 & 3
6. 2 & 3
7. 1 & 2
8. -
  • 3 ml LB + Amp 100; 30 °C
9. 1



Mimmi

Gel for Andreas colony-PCR

  • Make two gels, one 1% and one 0.8% agarose
  • Load samples in number order for Andreas to analyze



Over expression

  • Start up cultures
DNA
pEX.SOD
pEX.yCCS
pEX.SOD.his
pEX.his.SOD
    • 10ml LBAMP + 100µl old ON culture
  • At OD=0.6 (3h) add 1mM IPTG
    • Take sample at 0h and 3h
      • 1ml culture
      • spinn down cells
      • remove LB
      • add 100µl sample buffer
      • freeze
      • heat to 95°C for 10min

Nina

Colony PCR screen

I performed a PCR screen on the colonies from yesterday's transformation.

pEX Master Mix 15 tubes:

  • MgCl2 7.5 ul
  • phusion buffer 5X 75 ul
  • dNTP 7.5 ul
  • PrimerF 22.5 ul
  • PrimerR 22.5 ul
  • polymerase 7.5 ul
  • H2O 225 ul

Add 24 ul/PCR tube

pMa Master Mix 10 tubes:

  • MgCl2 5 ul
  • phusion buffer 5X 50 ul
  • dNTP 5 ul
  • PrimerF 15 ul
  • PrimerR 15 ul
  • polymerase 5 ul
  • H2O 150 ul

Add 24 ul/PCR tube

Bankvector C Master Mix 25 tubes:

  • MgCl2 12.5 ul
  • phusion buffer 5X 125 ul
  • dNTP 12.5 ul
  • PrimerF 37.5 ul
  • PrimerR 37.5 ul
  • polymerase 12.5 ul
  • H2O 375 ul

Add 24 ul/PCR tube





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/

Retrieved from "http://2010.igem.org/Team:Stockholm/28_September_2010"