Team:Stockholm/28 September 2010

From 2010.igem.org

(Difference between revisions)
(Andreas)
(Andreas)
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===Assembly and cloning===
===Assembly and cloning===
====Gel verification====
====Gel verification====
 +
[[image:ColPCR_pEX.nCPP.SH_28sep.png‎|200px|thumb|right|'''Colony PCR gel verification of pEX.nCPP&sdot;SOD&sdot;His constructs.'''<br />3 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
 +
[[image:ColPCR_nCPP.SH.Ry_28sep.png‎|200px|thumb|right|'''Colony PCR gel verification of nCPP&sdot;SOD&sdot;His.RBS.yCCS constructs.'''<br />3 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
 +
''Re-run of 27/9 colony PCRs.''
''Re-run of 27/9 colony PCRs.''
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'''Expected bands'''
'''Expected bands'''
-
#744 bp
+
#'''pEX.N-LMWP&sdot;SOD&sdot;His (K):''' 744 bp
-
#735 bp
+
#'''pEX.N-TAT&sdot;SOD&sdot;His:''' 735 bp
-
#765 bp
+
#'''pEX.N-Tra10&sdot;SOD&sdot;His:''' 765 bp
-
#744 bp
+
#'''pEX.N-LMWP&sdot;SOD&sdot;His (C):''' 744 bp
-
#1645 bp
+
#'''pSB1K3.N-LMWP&sdot;SOD&sdot;His.RBS.yCCS:''' 1645 bp
-
#1636 bp
+
#'''pSB1K3.N-TAT&sdot;SOD&sdot;His.RBS.yCCS:''' 1636 bp
-
#1666 bp
+
#'''pSB1K3.N-Tra10&sdot;SOD&sdot;His.RBS.yCCS:''' 1666 bp
-
#1645 bp
+
#'''pSB1C3.N-LMWP&sdot;SOD&sdot;His.RBS.yCCS:''' 1645 bp
-
#735 bp
+
#'''BL21 pEX.N-TAT&sdot;SOD&sdot;His 3:''' 735 bp
-
#735 bp
+
#'''BL21 pEX.N-TAT&sdot;SOD&sdot;His 4:''' 735 bp
'''Results'''<br />
'''Results'''<br />
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Set ON cultures for plasmid prep (constructs 1-8) and glycerol stock (9)
Set ON cultures for plasmid prep (constructs 1-8) and glycerol stock (9)
*5 ml LB + appr. antibiotic (Amp 100 or Km 50); 37 &deg;C, 220 rpm
*5 ml LB + appr. antibiotic (Amp 100 or Km 50); 37 &deg;C, 220 rpm
-
*#1
+
:1. 1
-
*#1
+
:2. 1
-
*#1
+
:3. 1
-
*#-
+
:4. -
-
*#2 & 3
+
:5. 2 & 3
-
*#2 & 3
+
:6. 2 & 3
-
*#1 & 2
+
:7. 1 & 2
-
*#-
+
:8. -
*3 ml LB + Amp 100; 30 &deg;C
*3 ml LB + Amp 100; 30 &deg;C
-
*#1
+
:9. 1

Revision as of 18:48, 30 September 2010


Contents

Andreas

Assembly and cloning

Gel verification

Colony PCR gel verification of pEX.nCPP⋅SOD⋅His constructs.
3 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.
Colony PCR gel verification of nCPP⋅SOD⋅His.RBS.yCCS constructs.
3 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

Re-run of 27/9 colony PCRs.

Gel 1
1 % agarose, 110 V

Gel 2
0.8 % agarose, 90 V

Expected bands

  1. pEX.N-LMWP⋅SOD⋅His (K): 744 bp
  2. pEX.N-TAT⋅SOD⋅His: 735 bp
  3. pEX.N-Tra10⋅SOD⋅His: 765 bp
  4. pEX.N-LMWP⋅SOD⋅His (C): 744 bp
  5. pSB1K3.N-LMWP⋅SOD⋅His.RBS.yCCS: 1645 bp
  6. pSB1K3.N-TAT⋅SOD⋅His.RBS.yCCS: 1636 bp
  7. pSB1K3.N-Tra10⋅SOD⋅His.RBS.yCCS: 1666 bp
  8. pSB1C3.N-LMWP⋅SOD⋅His.RBS.yCCS: 1645 bp
  9. BL21 pEX.N-TAT⋅SOD⋅His 3: 735 bp
  10. BL21 pEX.N-TAT⋅SOD⋅His 4: 735 bp

Results
Listing clones of seemingly correct sizes, thereby potentially correct constructs:

  1. 1
  2. 1 & 2
  3. 1 & 2
  4. No correct clones
  5. 2, 3 & 4
  6. 1, 2 & 3
  7. 1, 2, 3 & 4
  8. No correct clones
  9. 1 & 2
  10. No correct clones

For further verification constructs should be sent for sequencing. However, we will await the sequencing results from samples sent 27/9, as they were also used for building these new assemblies.

ON cultures

Set ON cultures for plasmid prep (constructs 1-8) and glycerol stock (9)

  • 5 ml LB + appr. antibiotic (Amp 100 or Km 50); 37 °C, 220 rpm
1. 1
2. 1
3. 1
4. -
5. 2 & 3
6. 2 & 3
7. 1 & 2
8. -
  • 3 ml LB + Amp 100; 30 °C
9. 1