Team:Stockholm/28 September 2010

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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

pEX.N-LMWP⋅SOD⋅His (K): 744 bp
pEX.N-TAT⋅SOD⋅His: 735 bp
pEX.N-Tra10⋅SOD⋅His: 765 bp
pEX.N-LMWP⋅SOD⋅His (C): 744 bp
pSB1K3.N-LMWP⋅SOD⋅His.RBS.yCCS: 1645 bp
pSB1K3.N-TAT⋅SOD⋅His.RBS.yCCS: 1636 bp
pSB1K3.N-Tra10⋅SOD⋅His.RBS.yCCS: 1666 bp
pSB1C3.N-LMWP⋅SOD⋅His.RBS.yCCS: 1645 bp
BL21 pEX.N-TAT⋅SOD⋅His 3: 735 bp
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
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 °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 LB_AMP + 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