Team:Stockholm/25 September 2010

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{{Stockholm/Top2}}
 
==Andreas==
==Andreas==
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#*pSB1C3.<u>N-LMWP&sdot;SOD&sdot;His</u> X+P
#*pSB1C3.<u>N-LMWP&sdot;SOD&sdot;His</u> X+P
#*<u>pEX</u>.SOD X+P
#*<u>pEX</u>.SOD X+P
-
#'''pSB1K3.N-LMWP&sdot;SOD&sdot;His'''
+
#'''pSB1K3.N-LMWP&sdot;SOD&sdot;His.RBS.yCCS'''
#*<u>pSB1K3.N-LMWP&sdot;SOD&sdot;His</u> S+P
#*<u>pSB1K3.N-LMWP&sdot;SOD&sdot;His</u> S+P
#*pSB1A2<u>.RBS.yCCS</u> X+P
#*pSB1A2<u>.RBS.yCCS</u> X+P
-
#'''pSB1K3.N-TAT&sdot;SOD&sdot;His'''
+
#'''pSB1K3.N-TAT&sdot;SOD&sdot;His.RBS.yCCS'''
#*<u>pSB1K3.N-TAT&sdot;SOD&sdot;His</u> S+P
#*<u>pSB1K3.N-TAT&sdot;SOD&sdot;His</u> S+P
#*pSB1A2<u>.RBS.yCCS</u> X+P
#*pSB1A2<u>.RBS.yCCS</u> X+P
-
#'''pSB1K3.N-Tra10&sdot;SOD&sdot;His'''
+
#'''pSB1K3.N-Tra10&sdot;SOD&sdot;His.RBS.yCCS'''
#*<u>pSB1K3.N-Tra10&sdot;SOD&sdot;His</u> S+P
#*<u>pSB1K3.N-Tra10&sdot;SOD&sdot;His</u> S+P
#*pSB1A2<u>.RBS.yCCS</u> X+P
#*pSB1A2<u>.RBS.yCCS</u> X+P
-
#'''pSB1C3.N-LMWP&sdot;SOD&sdot;His'''
+
#'''pSB1C3.N-LMWP&sdot;SOD&sdot;His.RBS.yCCS'''
#*<u>pSB1C3.N-LMWP&sdot;SOD&sdot;His</u> S+P
#*<u>pSB1C3.N-LMWP&sdot;SOD&sdot;His</u> S+P
#*pSB1A2<u>.RBS.yCCS</u> X+P
#*pSB1A2<u>.RBS.yCCS</u> X+P

Revision as of 17:17, 27 September 2010

Contents

Andreas

Cloning and assembly

Digestion

Further digestions in addition to the ones performed 24/9.

  pK.N-TAT⋅ SH (4) X+P pK.N-LMWP⋅ SH (4) X+P pK.N-LMWP⋅ SH (4) S+P
10X FastDigest buffer 2 2 2
DNA (1 μg) 8 4 4
dH2O 8 12 12
FD XbaI 1 1 0
FD SpeI 0 0 1
FD PstI 1 1 1
  20 μl 20 μl 20 μl
  • Incubation: 37 °C, 1 h
  • Inactivation: 80 °C, 30 min (including digestion samples from 24/9)

Ligation

Underlined sequences relevant for assembly

  1 2 3 4 5 6 7 8
10X T4 Ligase buffer 2 2 2 2 2 2 2 2
Vector DNA (50 ng) 1 1 1 1 1 1 1 1
Insert DNA 5 5 5 5 4 4 4 4
dH2O 11 11 11 11 12 12 12 12
T4 DNA ligase 1 1 1 1 1 1 1 1
  20 μl 20 μl 20 μl 20 μl 20 μl 20 μl 20 μl 20 μl
  1. pEX.N-LMWP⋅SOD⋅His (K)
    • pSB1K3.N-LMWP⋅SOD⋅His X+P
    • pEX.SOD X+P
  2. pEX.N-TAT⋅SOD⋅His
    • pSB1K3.N-TAT⋅SOD⋅His X+P
    • pEX.SOD X+P
  3. pEX.N-Tra10⋅SOD⋅His
    • pSB1K3.N-Tra10⋅SOD⋅His X+P
    • pEX.SOD X+P
  4. pEX.N-LMWP⋅SOD⋅His (C)
    • pSB1C3.N-LMWP⋅SOD⋅His X+P
    • pEX.SOD X+P
  5. pSB1K3.N-LMWP⋅SOD⋅His.RBS.yCCS
    • pSB1K3.N-LMWP⋅SOD⋅His S+P
    • pSB1A2.RBS.yCCS X+P
  6. pSB1K3.N-TAT⋅SOD⋅His.RBS.yCCS
    • pSB1K3.N-TAT⋅SOD⋅His S+P
    • pSB1A2.RBS.yCCS X+P
  7. pSB1K3.N-Tra10⋅SOD⋅His.RBS.yCCS
    • pSB1K3.N-Tra10⋅SOD⋅His S+P
    • pSB1A2.RBS.yCCS X+P
  8. pSB1C3.N-LMWP⋅SOD⋅His.RBS.yCCS
    • pSB1C3.N-LMWP⋅SOD⋅His S+P
    • pSB1A2.RBS.yCCS X+P

Transformation

  • Standard transformation according to protocol.
    • 1 μl ligation mix
    • Amp 100, Cm 25 or Km 50 plates
    • 50 μl IPTG (pEX constructs, Amp 100 plates)

Sequencing preparation

Prepared samples for sequencing to be sent on Monday.

Construct Primer Sample name
pEX.N-TAT⋅SOD⋅His 3 pEXf pEX.nTAT*SH3_pEXf
pEX.N-TAT⋅SOD⋅His 4 pEXf pEX.nTAT*SH4_pEXf
pSB1A2.RBS.yCCS 3 VF2 pA.RBS.yCCS3_VF2
pSB1A2.RBS.yCCS 4 VF2 pA.RBS.yCCS4_VF2
pSB1K3.N-TAT⋅SOD⋅His 4 VF2 pK.nTAT*SH4_VF2
pSB1K3.N-TAT⋅SOD⋅His 5 VF2 pK.nTAT*SH5_VF2
pSB1K3.N-Tra10⋅SOD⋅His 5 VF2 pK.nTra10*SH5_VF2
pSB1K3.N-LMWP⋅SOD⋅His 1 VF2 pK.nLMWP*SH1_VF2
pSB1C3.N-LMWP⋅SOD⋅His 1 VF2 pC.nLMWP*SH1_VF2
pSB1C3.N-LMWP⋅SOD⋅His 4 VF2 pC.nLMWP*SH4_VF2
pEX.SOD pEXf pEX.SOD_pEXf
pEX.yCCS 5 pEXf pEX.yCCS5_pEXf
pEX.SOD⋅His pEXf pEX.SH_pEXf
pEX.His⋅SOD pEXf pEX.HS_pEXf

BL21 transformation

Transformed BL21 cells with not-yet-verified pEX.N-TAT⋅SOD⋅His plasmid samples 3 & 4

  • Standard transformation according to protocol.
    • 1 μl ligation mix
      • 100 μl cell aliquot divided in 2x50 μl to lower number of colonies after transformation.
    • Amp 100 plates

Preparation of LB agar plates

Made new Cm (25 μg/ml) and Km (50 μg/ml) LB agar plates.

  • 10X Cm 25
  • 10X Km 50
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