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17/09/13
From 2013.igem.org
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| + | <div style="font-family:arial;padding:5px;border-radius:5px;border:5px solid #FF2800;"> | ||
| + | {| style="color:#87EA00;background-color:#FFFFFF;" cellpadding="2" cellspacing="2" border="0" bordercolor="#000000" width="100%" align="center" | ||
| + | !align="center"|[[Team:Leicester|Home]] | ||
| + | !align="center"|[[Team:Leicester/Team|Team]] | ||
| + | !align="center"|[https://igem.org/Team.cgi?year=2013&team_name=Leicester Official Team Profile] | ||
| + | !align="center"|[[Team:Leicester/Project|Project]] | ||
| + | !align="center"|[[Team:Leicester/Parts|Parts Submitted to the Registry]] | ||
| + | !align="center"|[[Team:Leicester/Modeling|Modeling]] | ||
| + | !align="center"|[[Team:Leicester/Notebook|Notebook]] | ||
| + | !align="center"|[[Team:Leicester/Safety|Safety]] | ||
| + | !align="center"|[[Team:Leicester/Attributions|Attributions]] | ||
| + | |} | ||
| + | </div> | ||
| + | |||
| + | |||
==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 13: | Line 28: | ||
==Digestion of new fusion PCR Tod genes== | ==Digestion of new fusion PCR Tod genes== | ||
*Digestion with EcorI-HF and PstI-HF using Cutsmart buffer | *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 | ||
| + | |- | ||
| + | |Tod X||49.5||1.89||1.99 | ||
| + | |- | ||
| + | |Tod F||33.9||1.98||1.68 | ||
| + | |- | ||
| + | |Tob B||37.4||1.82||1.70 | ||
| + | |} | ||
| + | *Volumes for double digest: | ||
{|border=1 | {|border=1 | ||
|Sample||DNA||Buffer||Water||EcorI-HF||PstI-HF | |Sample||DNA||Buffer||Water||EcorI-HF||PstI-HF | ||
| Line 22: | Line 48: | ||
|Tob B||14.7ul||3ul||11.3||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 |
|---|
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:
| Sample | Concentration ng/ul | 260/280 | 260/230 |
| Tod X | 49.5 | 1.89 | 1.99 |
| Tod F | 33.9 | 1.98 | 1.68 |
| Tob B | 37.4 | 1.82 | 1.70 |
- Volumes for double digest:
| 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:
| 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.
