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16/09/13
From 2013.igem.org
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(→Ligation of digested pSB1C3 backbone to Tod F, X and Tob G) |
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*Using Promega protocol to which changes were made | *Using Promega protocol to which changes were made | ||
{|border=1 | {|border=1 | ||
| - | |Reagents||Tod F||Tod X||Tob G||Positive control||Background control | + | |Reagents||Tod F||Tod X||Tob G||Positive control||Background control |
|- | |- | ||
|2x Rapid Ligation buffer||5ul||5ul||5ul||5ul||5ul | |2x Rapid Ligation buffer||5ul||5ul||5ul||5ul||5ul | ||
Latest revision as of 08:49, 17 September 2013
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Digesting pSB1C3 backbone
- Digesting for ligation with the already digested Tod operon PCR products
- Following the iGEM official protocol
- Using EcoRI-HF and PstI-HF
- Exluding the DpnI from the master mix and adjusting the volume of water accordingly
- Protocol can be found at: [http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones]
Ligation of digested pSB1C3 backbone to Tod F, X and Tob G
- Using Promega protocol to which changes were made
| Reagents | Tod F | Tod X | Tob G | Positive control | Background control |
| 2x Rapid Ligation buffer | 5ul | 5ul | 5ul | 5ul | 5ul |
| pSB1C3 digest (50ng) | 2ul | 2ul | 2ul | 2ul | 2ul |
| PCR product | 3.3ul | 3ul | 3.8ul | - | - |
| Control insert DNA | - | - | - | 2ul | - |
| T4 DNA ligase | 1ul | 1ul | 1ul | 1ul | 1ul |
| Deionized water for final volume of 10ul | - | - | - | - | 1ul |
- Mix reactions by pipetting. Incubate the reactions for 1h at room temperature.
- Control insert DNA from the transformation efficiency kit provided by iGEM
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 1.5 hours at 37C with shaking.
Digestion of the TOD genes PCR products
The PCR products of TODX, TODF and TOBG were digested with the enzymes EcoRI-HF and PstI-HF.
Protocol
| Sample | DNA volume (ul) | cutsmart buffer (ul) | EcoRI (ul) | PstI (ul) | dH2O (ul) |
| TODX | 8.7 | 2 | 0.5 | 0.5 | 8.3 |
| TODF | 8.3 | 2 | 0.5 | 0.5 | 8.7 |
| TOBG | 8 | 2 | 0.5 | 0.5 | 9 |
- The samples were incubated for 1 hour at 37C.
- Heat kill the enzymes at 80C for 20 minutes.
Running the backbone and RFP on gel
- on 1% gel to check if the gel extraction worked.
- gel shows that extraction worked
- 2kb band representing the backbone is visible as well as 1kb representing the RFP
Digestion of the pGEM plasmid gel extracted on the 13/09/13
The samples were digested with the enzymes EcoRI-HF and PstI-HF
Master Mix for 8 reactions
- 16 ul of cutsmart buffer
- 4 ul of EcoRI
- 4 ul of PstI
- 88 ul of dH2O
- 14 ul of master mix was added to each reaction
