Team:Stockholm/23 September 2010

From 2010.igem.org

(Difference between revisions)
(New page: {{Stockholm/Top2}} ==Andreas==)
(→Andreas)
Line 1: Line 1:
{{Stockholm/Top2}}
{{Stockholm/Top2}}
==Andreas==
==Andreas==
 +
 +
===BL21(DE3) clone verification===
 +
Attempted to verify BL21 clones from 21/9 transformations for correct plasmid (pEX) and insert.
 +
 +
====Colony PCR====
 +
*pEX.SOD: 1 & 2
 +
*pEX.SOD⋅His (pEX.SH): 1 & 2
 +
*pEX.His⋅SOD (pEX.HS): 1 & 2
 +
*pEX.yCCS 5: 1 & 2
 +
*pEX.His⋅SOD plasmid (PC)
 +
 +
PCR settings according to standard colony PCR protocol.
 +
*Elongation time: 1:30
 +
 +
====Gel verification====
 +
[[image:ColPCR_BL21_clones_23sep.png|200px|thumb|right|'''Colony PCR gel verification of BL21 clones transformed with pEX.SOD, pEX.SOD&sdot;His, pEX.His&sdot;SOD or pEX.yCCS 5.'''<br />4 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
 +
 +
1 % agarose, 120 V
 +
 +
'''Expected bands'''
 +
*pEX.SOD: 678 bp
 +
*pEX.SOD&sdot;His (pEX.SH): 702 bp
 +
*pEX.His&sdot;SOD (pEX.HS): 702 bp
 +
*pEX.yCCS: 963 bp
 +
 +
'''Results'''<br />
 +
Very weak but correct-sized bands for all clones but pEX.yCCS 5 #1. Clones verified.
 +
 +
====ON cultures====
 +
One verified clone of each BL21 construct was chosen for preparation of glycerol stocks:
 +
*3 ml LB + 100 Amp; 30 &deg;C
 +
**pEX.SOD: clone 1
 +
**pEX.SOD&sdot;His (pEX.SH): clone 1
 +
**pEX.His&sdot;SOD (pEX.HS): clone 1
 +
**pEX.yCCS: clone 2
 +
 +
===Plasmid prep===
 +
''From 22/9 ON cultures''
 +
 +
Using Omega E.Z.N.A. Plasmid Miniprep kit I.
 +
 +
No growth in cultures pSB1K3.N-LMWP&sdot;SOD&sdot;His and pEX.N-TAT&sdot;SOD&sdot;His 4.
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="3"|DNA concentration
 +
|-
 +
!Sample
 +
!width="60"|Conc [ng/&mu;l]
 +
!width="60"|A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pSB1C3.N-LMWP&sdot;SOD&sdot;His 1
 +
|align="center"|35.06
 +
|align="center"|1.68
 +
|-
 +
|pSB1C3.N-LMWP&sdot;SOD&sdot;His 4
 +
|align="center"|46.85
 +
|align="center"|1.68
 +
|-
 +
|pSB1A2.RBS.yCCS 3
 +
|align="center"|39.64
 +
|align="center"|1.59
 +
|-
 +
|pSB1A2.RBS.yCCS 4
 +
|align="center"|87.01
 +
|align="center"|1.49
 +
|-
 +
|pEX.N-TAT&sdot;SOD&sdot;His 3
 +
|align="center"|66.22
 +
|align="center"|1.67
 +
|}
 +
 +
Very low DNA concentrations and low purity. Possibly something wrong with the plasmid miniprep kit. New plasmid preps will be prepared tomorrow, using brand new kit solutions.
 +
 +
====ON cultures====
 +
*5 ml LB + antibiotic (100 Amp, 50 Km or 25 Cm); 37 &deg;C 200 rpm
 +
**pEX.N-TAT&sdot;SOD&sdot;His 3 (100 Amp)
 +
**pEX.N-TAT&sdot;SOD&sdot;His 4 (100 Amp)
 +
**pSB1C3.N-LMWP&sdot;SOD&sdot;His 1 (25 Cm)
 +
**pSB1C3.N-LMWP&sdot;SOD&sdot;His 4 (25 Cm)
 +
**pSB1A2.RBS.yCCS 3 (100 Amp)
 +
**pSB1A2.RBS.yCCS 4 (100 Amp)
 +
**pSB1K3.N-LMWP&sdot;SOD&sdot;His 1 (50 Km)
 +
**pSB1K3.N-TAT&sdot;SOD&sdot;His 4 (50 Km)
 +
**pSB1K3.N-TAT&sdot;SOD&sdot;His 5 (50 Km)
 +
**pSB1K3.N-Tra10&sdot;SOD&sdot;His 5 (50 Km)

Revision as of 21:06, 23 September 2010

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

↳   Notebook Safety Protocols


Contents

Andreas

BL21(DE3) clone verification

Attempted to verify BL21 clones from 21/9 transformations for correct plasmid (pEX) and insert.

Colony PCR

  • pEX.SOD: 1 & 2
  • pEX.SOD⋅His (pEX.SH): 1 & 2
  • pEX.His⋅SOD (pEX.HS): 1 & 2
  • pEX.yCCS 5: 1 & 2
  • pEX.His⋅SOD plasmid (PC)

PCR settings according to standard colony PCR protocol.

  • Elongation time: 1:30

Gel verification

Colony PCR gel verification of BL21 clones transformed with pEX.SOD, pEX.SOD⋅His, pEX.His⋅SOD or pEX.yCCS 5.
4 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

1 % agarose, 120 V

Expected bands

  • pEX.SOD: 678 bp
  • pEX.SOD⋅His (pEX.SH): 702 bp
  • pEX.His⋅SOD (pEX.HS): 702 bp
  • pEX.yCCS: 963 bp

Results
Very weak but correct-sized bands for all clones but pEX.yCCS 5 #1. Clones verified.

ON cultures

One verified clone of each BL21 construct was chosen for preparation of glycerol stocks:

  • 3 ml LB + 100 Amp; 30 °C
    • pEX.SOD: clone 1
    • pEX.SOD⋅His (pEX.SH): clone 1
    • pEX.His⋅SOD (pEX.HS): clone 1
    • pEX.yCCS: clone 2

Plasmid prep

From 22/9 ON cultures

Using Omega E.Z.N.A. Plasmid Miniprep kit I.

No growth in cultures pSB1K3.N-LMWP⋅SOD⋅His and pEX.N-TAT⋅SOD⋅His 4.

DNA concentration
Sample Conc [ng/μl] A260/A280
pSB1C3.N-LMWP⋅SOD⋅His 1 35.06 1.68
pSB1C3.N-LMWP⋅SOD⋅His 4 46.85 1.68
pSB1A2.RBS.yCCS 3 39.64 1.59
pSB1A2.RBS.yCCS 4 87.01 1.49
pEX.N-TAT⋅SOD⋅His 3 66.22 1.67

Very low DNA concentrations and low purity. Possibly something wrong with the plasmid miniprep kit. New plasmid preps will be prepared tomorrow, using brand new kit solutions.

ON cultures

  • 5 ml LB + antibiotic (100 Amp, 50 Km or 25 Cm); 37 °C 200 rpm
    • pEX.N-TAT⋅SOD⋅His 3 (100 Amp)
    • pEX.N-TAT⋅SOD⋅His 4 (100 Amp)
    • pSB1C3.N-LMWP⋅SOD⋅His 1 (25 Cm)
    • pSB1C3.N-LMWP⋅SOD⋅His 4 (25 Cm)
    • pSB1A2.RBS.yCCS 3 (100 Amp)
    • pSB1A2.RBS.yCCS 4 (100 Amp)
    • pSB1K3.N-LMWP⋅SOD⋅His 1 (50 Km)
    • pSB1K3.N-TAT⋅SOD⋅His 4 (50 Km)
    • pSB1K3.N-TAT⋅SOD⋅His 5 (50 Km)
    • pSB1K3.N-Tra10⋅SOD⋅His 5 (50 Km)
Retrieved from "http://2010.igem.org/Team:Stockholm/23_September_2010"