17/09/13

From 2013.igem.org

(Difference between revisions)
(→Digestion of new fusion PCR Tod genes)
(→Digestion of new fusion PCR Tod genes)
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|-
|-
|Tob B||14.7ul||3ul||11.3||0.5||0.5
|Tob B||14.7ul||3ul||11.3||0.5||0.5
 +
|}
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*Incubation at 37C for 30min
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*Heat kill at 80C for 20min
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 +
==Ligation of Tod genes and pSB1C3 backbone==
 +
*For background control, just the pSB1C3 backbone was used
 +
*For positive control, RFP plasmid from transformation efficiency kit provide by iGEM was used
 +
*Quick stick ligase was used and a different protocol from previous day
 +
*Bioline Quick Stick Ligase protocol:
 +
***Combine the vector and the insert in the appropriate ratio to make up no more than 100ng of DNA
 +
***Adjust volume to 14ul with ddH2O
 +
***Add 1ul of QS ligase
 +
***Add 5ul of 4x QS Buffer (vortex before use)
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**Mix thoroughly before pipetting
 +
**Incubate at room temperature for 5min
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*The volumes for ligation:
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{|
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|Sample||DNA ul||Vector(pSB1C3) ul||QS ligase ul||Buffer ul||H20 ul
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|-
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|Tod X||0.9||2||1||5||5.1
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|-
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|Tod F||0.8||2||1||5||5.2
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|-
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|Tob B||1||2||1||5||5
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|-
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|+ control||1||2||1||5||5
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|-
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|Background control||0||2||1||5||6
|}
|}

Revision as of 10:36, 18 September 2013

Contents

Results from previous day

  • Background control plate had growth, which was not expected. This suggests that pSB1C3 backbone religated.
  • It was also thought that the Tod genes used were not from the PCR fusion experiment.
  • Same experiment done again, with a new set of Tod genes from a PCR fusion.
  • For digestion of backbone, already digested backbone from 01/08/2013 was used, which was digested with EcoRI and PstI.

Incubation of bacteria from the plates of previous day for mini prep

  • 12 single colonies were picked from each plate and put into 15ml of broth
  • 36 tubes were left incubating overnight at 37C

Sending pGEM-T vector with Tod insert clones to sequencing by PNACL

Digestion of new fusion PCR Tod genes

  • Digestion with EcorI-HF and PstI-HF using Cutsmart buffer
  • 500ng of DNA was digested from the following Tod DNA concentrations:
SampleConcentration ng/ul260/280260/230
Tod X49.51.891.99
Tod F33.91.981.68
Tob B37.41.821.70
  • Volumes for double digest:
SampleDNABufferWaterEcorI-HFPstI-HF
Tod X10.1ul3ul15.90.50.5
Tod F13.4ul3ul12.60.50.5
Tob B14.7ul3ul11.30.50.5
  • Incubation at 37C for 30min
  • Heat kill at 80C for 20min

Ligation of Tod genes and pSB1C3 backbone

  • For background control, just the pSB1C3 backbone was used
  • For positive control, RFP plasmid from transformation efficiency kit provide by iGEM was used
  • Quick stick ligase was used and a different protocol from previous day
  • Bioline Quick Stick Ligase protocol:
      • Combine the vector and the insert in the appropriate ratio to make up no more than 100ng of DNA
      • Adjust volume to 14ul with ddH2O
      • Add 1ul of QS ligase
      • Add 5ul of 4x QS Buffer (vortex before use)
    • Mix thoroughly before pipetting
    • Incubate at room temperature for 5min
  • The volumes for ligation:
SampleDNA ulVector(pSB1C3) ulQS ligase ulBuffer ulH20 ul
Tod X0.92155.1
Tod F0.82155.2
Tob B12155
control12155
Background control02156
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