Team:Stockholm/28 September 2010

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Revision as of 18:48, 30 September 2010

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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