Team:Stockholm/2 September 2010

From 2010.igem.org

(Difference between revisions)
(→Transformation results)
(→Andreas)
Line 18: Line 18:
====Gel verification====
====Gel verification====
-
 
+
[[image:Col-PCR His.SOD 2sep.png|200px|right|thumb|'''Gel verification of pMA.His&sdot;SOD colony PCR.'''<br />3 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda;=O'GeneRuler 1 kb DNA ladder]]
1 % agarose
1 % agarose
 +
 +
'''Expected bands'''
 +
*pMA.His&sdot;SOD: 831 bp
 +
*pMA.His: 366 bp
 +
 +
'''Results'''<br />
 +
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 &deg;C, 225 rpm), and glycerol stock preparation (3 ml LB + 100 Amp, 30 &deg;C)
 +
 +
===Plasmid prep===
 +
''From 1/9 ON cultures''
 +
 +
Elution: 70 &mu;l x2
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="3"|DNA concentrations
 +
|-
 +
!Sample
 +
!Conc. [ng/&mu;l]
 +
! A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pSB1C3.m-yCCS 1
 +
|align="center"|143.4
 +
|align="center"|1.91
 +
|-
 +
|pMA.His
 +
|align="center"|175.9
 +
|align="center"|1.90
 +
|-
 +
|pEX.RFP
 +
|align="center"|44.95
 +
|align="center"|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 &mu;l 0.1 M IPTG had first been spread. Plate incubated ON in 37 &deg;C.

Revision as of 10:28, 6 September 2010

Home Project Idea Lab Work Results Modelling Team       Follow us on and        2010.igem.org

↳   Notebook Safety Protocols


Contents

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

Gel verification of pMA.His⋅SOD colony PCR.
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.

Retrieved from "http://2010.igem.org/Team:Stockholm/2_September_2010"