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From 2010.igem.org

Contents 1 Andreas 1.1 Preparation of Top10 chemically competent cells 1.2 Cloning of N-CPPs into pSB1C3 1.2.1 Transformations 1.2.2 Sequencing 2 Mimmi 2.1 MITF-M 2.1.1 Site-Directed Mutagenesis control 2.1.2 Gel 2.2 RBS 2.2.1 plasmid prep.

Andreas

Preparation of Top10 chemically competent cells

None of the clones streaked onto the Amp 100 plate (11/9) grew, indicating no AmpR contamination in cells. One of the ON cultures were therefore selected for preparation of competent cells.

Procedures according to protocol. Growth conditions changed to 25 °C, 220 rpm (OD₆₀₀ reached after ≈6 h).

A 100 μl aliquot was divided and spread onto Amp 100, Cm 25 and Km 50 plates to verify that they were not contaminated.

Cloning of N-CPPs into pSB1C3

Sequencing results from 8/9 returned.

• pSB1C3.nCPP 3 (failed)
• pSB1C3.nCPP 4 (fasta)
• pSB1C3.nCPP 6 (fasta)
• pSB1C3.nCPP 7 (fasta)
• pSB1C3.nCPP 8 (fasta)

Blastn alignments against N-Tra10, N-TAT and N-LMWP indicated successful cloning of N-Tra10 (clone 7) and N-TAT (clone 4).

Transformations

Since pSB1C3.N-TAT and pSB1C3.N-Tra10 colony samples were accidentally discarded, prepared plasmids were used to transform new cells in order to prepare glycerol stocks.

Standard transformation with 1 μl plasmid DNA.

• pSB1C3.N-TAT
• pSB1C3.N-Tra10

Sequencing

DNA concentrations of 11/9 plasmid preps were measured by Mimmi and samples were sent for sequencing for isolation of N-LMWP.

• pSB1C3.nCCP 2: ABS0045 B92
• pSB1C3.nCCP 3: ABS0045 B93
• pSB1C3.nCCP 5: ABS0045 B94
• pSB1C3.nCCP 8: ABS0045 B95
• pSB1C3.nCCP 9: ABS0045 B96
• pSB1C3.nCCP 10: ABS0045 B97
• pSB1C3.nCCP 11: ABS0045 B98
• pSB1C3.nCCP 12: ABS0045 B99

Mimmi

MITF-M

Site-Directed Mutagenesis control

mix	(µl)		Conditions
DNA	20		time	°C
10x buffer	3		3h	37
sH2O	5
XbaI	1
AgeI	1
tot	30µl

Gel

well	sample
1	ladder
2	MITF-col 1
3	MITF-col 1 cut X+A
4	MITF-col 2
5	MITF-col 2 cut X+A
6	MITF-col 3
7	MITF-col 3 cut X+A
8	MITF-col 4
9	MITF-col 4 cut X+A
10	MITF-M
11	MITF-M cut X+A

• Should have saved more original MITF (stupid!)
  • Make new MITF-M

RBS

plasmid prep.

• Follow original protocol
  • Wash 2 times with DNA wash buffer
  • Eluate in 50µl
  • Eluate 2 times with the same sH₂O

DNA conc. RBS34a ~42ng/µl -> ~60ng/µl RBS34b ~41ng/µl -> ~60ng/µl