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From 2010.igem.org

Contents 1 Andreas 1.1 His⋅SOD cloning into pMA 1.1.1 Transformation results 1.1.2 Colony PCR 1.1.3 Gel verification 1.1.4 ON cultures 1.2 Plasmid prep 1.3 Transfer of m-yCCS into pEX 2 Mimmi 2.1 MITF-M 2.1.1 Transformation

Andreas

His⋅SOD cloning into pMA

Transformation results

From 1/9 transformations

Much growth on both Top10 and DH5α plates, with Top10 being slightly more competent.

Colony PCR

Three clones selected for colony PCR. Standard colony PCR procedures, see protocol. Elongation time: 1 min.

• HS1
• HS2
• HS3
• Pos. control (PC): pMA.His

Gel verification

1 % agarose

Expected bands

• pMA.His⋅SOD: 831 bp
• pMA.His: 366 bp

Results
Clone HS 3 seems correct, while HS 1 and HS 2 are too short.

ON cultures

Set ON cultures of HS 3 for plasmid prep (5 ml LB + 100 Amp, 37 °C, 225 rpm), and glycerol stock preparation (3 ml LB + 100 Amp, 30 °C)

Plasmid prep

From 1/9 ON cultures

Elution: 70 μl x2

DNA concentrations
Sample	Conc. [ng/μl]	A260/A280
pSB1C3.m-yCCS 1	143.4	1.91
pMA.His	175.9	1.90
pEX.RFP	44.95	1.88

Transfer of m-yCCS into pEX

Since no IPTG was spread on agar plate prior to plating (1/9), there was no expression of RFP, i.e. no red colonies. 12 colonies were therefore restreaked onto a new Amp 100 LB agar plate to which 50 μl 0.1 M IPTG had first been spread. Plate incubated ON in 37 °C.

Mimmi

MITF-M

Transformation

• Follow original protocol
  • 1µl plasmid + 2µl dephosphorylated plasmid