"

From 2010.igem.org

Contents 1 Andreas 1.1 Assembly and cloning 1.1.1 Gel verification 1.1.2 ON cultures 2 Mimmi 2.1 Gel for Andreas colony-PCR 2.2 Over expression 3 Nina 3.1 Colony PCR screen

Andreas

Assembly and cloning

Gel verification

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 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

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