17/09/13

From 2013.igem.org

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{| style="color:#87EA00;background-color:#FFFFFF;" cellpadding="2" cellspacing="2" border="0" bordercolor="#000000" width="100%" align="center"
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!align="center"|[[Team:Leicester|Home]]
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!align="center"|[[Team:Leicester/Team|Team]]
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!align="center"|[https://igem.org/Team.cgi?year=2013&team_name=Leicester Official Team Profile]
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!align="center"|[[Team:Leicester/Project|Project]]
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!align="center"|[[Team:Leicester/Parts|Parts Submitted to the Registry]]
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!align="center"|[[Team:Leicester/Modeling|Modeling]]
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!align="center"|[[Team:Leicester/Notebook|Notebook]]
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!align="center"|[[Team:Leicester/Safety|Safety]]
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!align="center"|[[Team:Leicester/Attributions|Attributions]]
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|}
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</div>
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==Results from previous day==
==Results from previous day==
*Background control plate had growth, which was not expected. This suggests that pSB1C3 backbone religated.  
*Background control plate had growth, which was not expected. This suggests that pSB1C3 backbone religated.  
Line 5: Line 20:
*For digestion of backbone, already digested backbone from 01/08/2013 was used, which was digested with EcoRI and PstI.
*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:
 +
{|border=1
 +
|Sample||Concentration ng/ul||260/280||260/230
 +
|-
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|Tod X||49.5||1.89||1.99
 +
|-
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|Tod F||33.9||1.98||1.68
 +
|-
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|Tob B||37.4||1.82||1.70
 +
|}
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*Volumes for double digest:
 +
{|border=1
 +
|Sample||DNA||Buffer||Water||EcorI-HF||PstI-HF
 +
|-
 +
|Tod X||10.1ul||3ul||15.9||0.5||0.5
 +
|-
 +
|Tod F||13.4ul||3ul||12.6||0.5||0.5
 +
|-
 +
|Tob B||14.7ul||3ul||11.3||0.5||0.5
 +
|}
 +
*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:
 +
{|border=1
 +
|Sample||DNA ul||Vector(pSB1C3) ul||QS ligase ul||Buffer ul||H20 ul
 +
|-
 +
|Tod X||0.9||2||1||5||5.1
 +
|-
 +
|Tod F||0.8||2||1||5||5.2
 +
|-
 +
|Tob B||1||2||1||5||5
 +
|-
 +
|Positive control||1||2||1||5||5
 +
|-
 +
|Background control||0||2||1||5||6
 +
|}
 +
 
 +
==Transformation of TOD genes ligations with the digested pSB1C3 using the pGEM-T Vector System I-A3600 (changes were made to the protocol)==
 +
 
 +
*Centrifuge the ligations reactions briefly.
 +
*Add 5ul of each ligation reaction to a sterile 1.5ml
 +
*Thaw the competent cells on ice. Mix the cells by gently flicking the tube.
 +
*Carefully transfer 50ul of the competent cells to the ligation reaction tubes.
 +
*Incubate the tubes on ice for 20 minutes
 +
*Heat-shock the cells for 45-50 seconds in a water bath at exactly 42C degrees. Do not Shake. Immediately return the tubes to ice for 2 minutes.
 +
*Add 900ul of room temperature SOC medium to the ligation reaction transformations. incubate for 1 hour at 37C with shaking.
 +
 
 +
==Plating out transformants on chloroamphenicol plates==
 +
*Plating out a total of 10 plates, 2 for each sample
 +
*20ul and 200ul were plated from each sample
 +
*Plates were left over night at 37C.

Latest revision as of 08:58, 19 September 2013

Home Team Official Team Profile Project Parts Submitted to the Registry Modeling Notebook Safety Attributions


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
Positive control12155
Background control02156

Transformation of TOD genes ligations with the digested pSB1C3 using the pGEM-T Vector System I-A3600 (changes were made to the protocol)

  • Centrifuge the ligations reactions briefly.
  • Add 5ul of each ligation reaction to a sterile 1.5ml
  • Thaw the competent cells on ice. Mix the cells by gently flicking the tube.
  • Carefully transfer 50ul of the competent cells to the ligation reaction tubes.
  • Incubate the tubes on ice for 20 minutes
  • Heat-shock the cells for 45-50 seconds in a water bath at exactly 42C degrees. Do not Shake. Immediately return the tubes to ice for 2 minutes.
  • Add 900ul of room temperature SOC medium to the ligation reaction transformations. incubate for 1 hour at 37C with shaking.

Plating out transformants on chloroamphenicol plates

  • Plating out a total of 10 plates, 2 for each sample
  • 20ul and 200ul were plated from each sample
  • Plates were left over night at 37C.
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