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<div class = "col-md-12"> | <div class = "col-md-12"> | ||
− | <h1 style="text-align:center; font-size: 50px;"> <font face= "Poiret One"> | + | <h1 style="text-align:center; font-size: 50px;"> <font face= "Poiret One">Proximity dependent ligation assay #1 |
</h2> | </h2> | ||
<br> | <br> | ||
− | + | ||
− | 1) | + | <p style="text-align:center; font-size:20px;"><font face="verdana"> |
+ | 1) Dilute all probe oligos to 100uM and all connector oligos to 200uM in nuclease free water. | ||
</font><br><hr></p> | </font><br><hr></p> | ||
<p style="text-align:center; font-size:20px;"><font face="verdana"> | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
− | 2) | + | 2) In PCR tubes mix the following 6 reactions: |
</font><br><hr></p> | </font><br><hr></p> | ||
<p style="text-align:center; font-size:20px;"><font face="verdana"> | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
− | + | -----A) 2uL 10X DNA Ligase buffer, 2uL NEB DNA ligase, 2uL probe 1, 2uL probe 2, 1uL connector 1C, 11uL nuclease free water | |
</font><br><hr></p> | </font><br><hr></p> | ||
<p style="text-align:center; font-size:20px;"><font face="verdana"> | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
− | + | -----B) 2uL 10X DNA Ligase buffer, 2uL NEB DNA ligase, 2uL probe 1, 2uL probe 2, 1uL connector 2C, 11uL nuclease free water | |
+ | </font><br><hr></p> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | -----C) 2uL 10X DNA Ligase buffer, 2uL NEB DNA ligase, 2uL probe 1, 2uL probe 2, 1uL connector 3C, 11uL nuclease free water | ||
+ | </font><br><hr></p> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | -----D) 2uL 10X DNA Ligase buffer, 2uL NEB DNA ligase, 2uL probe 1, 2uL probe 2, 1uL connector 1C, 0.979uL Thrombin, 10.02uL nuclease free water | ||
+ | </font><br><hr></p> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | -----E) 2uL 10X DNA Ligase buffer, 2uL NEB DNA ligase, 2uL probe 1, 2uL probe 2, 1uL connector 2C, 0.979uL Thrombin, 10.02uL nuclease free water | ||
+ | </font><br><hr></p> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | -----F) 2uL 10X DNA Ligase buffer, 2uL NEB DNA ligase, 2uL probe 1, 2uL probe 2, 1uL connector 3C, 0.979uL Thrombin, 10.02uL nuclease free water | ||
+ | </font><br><hr></p> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | 3) Incubate reactions at 37C for 2 hours, then heat inactivate at 70C for 10 minutes | ||
+ | </font><br><hr></p> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | 4) Add 6X loading dye to each sample. | ||
+ | </font><br><hr></p> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | 5) Load the 6 reactions into a 1.75% agarose gel, along with 1kb ladder and 105.5ng of probe 1 in its own lane as a control. | ||
+ | </font><br><hr></p> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | 6) Run the gel. | ||
</font><br><hr></p> | </font><br><hr></p> | ||
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− | |||
</div> | </div> | ||
</div> | </div> | ||
+ | <div class= "row"> | ||
+ | <div class = "blue"> | ||
+ | <div class = "col-md-12"> | ||
+ | <h1 style="text-align:center; font-size: 50px;"> <font face= "Poiret One">Proximity dependent ligation assay #2 | ||
+ | </h2> | ||
+ | <br> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | Same as previous protocol, but incubate reactions at 35C for 9 hours. | ||
+ | </font><br><hr></p> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class= "row"> | ||
+ | <div class = "maize"> | ||
+ | <div class = "col-md-12"> | ||
+ | <h1 style="text-align:center; font-size: 50px;"> <font face= "Poiret One">Proximity dependent ligation assay #3 | ||
+ | </h2> | ||
+ | <br> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | Same as previous protocol, but incubate reactions at 25C for 4 hours and use 2% agasose gel. | ||
+ | </font><br><hr></p> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class= "row"> | ||
+ | <div class = "maize"> | ||
+ | <div class = "col-md-12"> | ||
+ | <h1 style="text-align:center; font-size: 50px;"> <font face= "Poiret One">Proximity dependent ligation assay #4 | ||
+ | </h2> | ||
+ | <br> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | Same as previous protocol, but make two sets of the reactions. Incubate one set of reactions at 20C and the other set at 37C for 4 hours and use 2% agasose gel. | ||
+ | </font><br><hr></p> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="row"> | ||
+ | <div class = "maize"> | ||
+ | <div class="col-md-12"> | ||
+ | <h1 style="text-align:center; font-size: 50px;"><font face= "Poiret One">NEB Q5 Site-directed Mutagenesis </h2> | ||
+ | |||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | 1) Assemble the following reagents in a thin-walled PCR tube: 12.5uL Q5 Hot Start High-Fidelity 2X Master Mix, 1.25uL 10 μM Forward Primer, 1.25uL 10 μM Reverse Primer, 1uL Template DNA (1-25 ng/uL), 9uL nuclease free water. | ||
+ | </font><br><hr></p> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | 2) Mix reagents completely, then transfer to a thermocycler. | ||
+ | </font><br><hr></p> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | 3) Perform the following cycling conditions: 30s @ 98C, 25x[10s @98C, 10-30s @ 50-72C (use NEB annealing calculator), 25s/kb @72C], 2mins @72C, hold at 4C. | ||
+ | </font><br><hr></p> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | 4) Assemble the following reagents: 1uL PCR product, 5uL 2X KLD Reaction Buffer, 1uL 10X KLD Enzyme Mix, 3ul Nuclease-free Water. | ||
+ | </font><br><hr></p> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | 5) Mix well by pipetting up and down and incubate at room temperature for 5 minutes. | ||
+ | </font><br><hr></p> | ||
+ | <p style="text-align:center; font-size:20px;"><font face="verdana"> | ||
+ | 6) Transform into chemically competent cells. | ||
+ | </font><br><hr></p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
</body> | </body> | ||
</html> | </html> |
Latest revision as of 03:45, 20 October 2016
Experimental Flow
Below we have outlined the path our project took, from the initial cloning through experimentation and submission.
Isolated LacZ from iGem part BBa_K564012
1) Rehydrated DNA from distribution kit
2) Transformed into DH5a chemically competent cells
3) Extracted plasmid
4) PCR mutagenesis to engineer desired cut sites around LacZ
Transferring LacZ into pET28
1) Digested pET28 and new version of BBa_K564012 (with added cut sites)
2) Ligated together
3) Submitted for sequencing to confirm
4) Transformed into new DH5a chemically competent cells
Creation of delta-M15 mutant
1) Deletion of segment including alpha fragment of LacZ using NEB Q5 site directed mutagenesis kit
2) Submitted for sequencing to confirm successful deletion
Proximity dependent ligation assay
1) Probe segments and bridge segments mixed with T4 ligase in presence or absence of thrombin
2) Gel electropheresis to detect ligation
Cloned delta-M15 mutant into submission vector
1) Digested linearized pSB1C3 with our mutant
2) Ligated together
3) Transformed into new DH5a chemically competent cells
5) Extracted DNA
6) Dried, packaged, shipped
3) Transformed into new DH5a chemically competent cells
Protocols
Below are the protocols we used for al stages of our project. We wrote them to be as easy to follow as possible, to accommodate new team members with little lab experience. We hope future teams find these highly detailed versions of common protocols useful.