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Team:Stockholm/2 September 2010
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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
3 μl λ; 5 μl sample.
λ=O'GeneRuler 1 kb DNA ladder
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
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