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Team:Grinnell/Notebook/Protocols
From 2011.igem.org
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<li> Run PCR tubes on following thermal cycle program</li> | <li> Run PCR tubes on following thermal cycle program</li> | ||
| - | |||
<table frame="void" rules="none"> | <table frame="void" rules="none"> | ||
<tr><th>Step</th><th>Temperature (°C)</th><th>Time (sec)</th></tr> | <tr><th>Step</th><th>Temperature (°C)</th><th>Time (sec)</th></tr> | ||
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<tr><td>9</td><td>4</td><td>hold</td></tr></table> | <tr><td>9</td><td>4</td><td>hold</td></tr></table> | ||
</br> | </br> | ||
| - | |||
<table frame="void" rules="none"> | <table frame="void" rules="none"> | ||
| - | <tr><th> | + | <tr><th>DNA sample</th><th>Temperature used in step 3(°C)</th><th>Temperature used in step 6(°C)</th><th>Time used for extention steps(°C)</th></tr> |
| - | + | <tr><td>rsaA</td><td>60</td><td>72.1</td><td>30</td></tr> | |
| - | <tr><td> | + | <tr><td>esp</td><td>41.2</td><td>68.4</td><td>45</td></tr> |
| - | + | <tr><td>rsaA Promotor</td><td>61.6</td><td>73.4</td><td>30</td></tr> | |
| - | <tr><td> | + | <tr><td>Pxyl</td><td>49.2</td><td>71.4</td><td>30</td></tr> |
| - | + | </table> | |
| - | <tr><td> | + | |
| - | + | ||
| - | <tr><td> | + | |
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<li>Take amplified DNA from the clear liquid layer on the top.</li> | <li>Take amplified DNA from the clear liquid layer on the top.</li> | ||
</ol> | </ol> | ||
Revision as of 20:25, 13 June 2011
Competent Cells
- Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5 x 10^8 cells/mL, OD600 = 0.3-0.5).
- Chill cells on ice for 15-120min.
- Pellet cells in a prechilled sterile centrifuge tube by centrifugation at 5-8krpm for 5min at 4° C. Discard supernatant.
- Completely resuspend cells in 20mL cold 100mM CaCl2 (10% glycerol), and incubate on ice for 3hr.
- Harvest cells by cetrifugation. Discard supernatant.
- Gently resuspend cells in 0.5mL cold 100mL CaCl2 (10% glycerol). Incubate on ice for at least 1hr and freeze at -80° C.
Plasmid Transformation
- Thaw 100μL aliquots of competent cells on ice.
- Add 10μL DNA to cells.
- Incubate tubes on ice for 30min.
- Incubate tubes at 42° C for 90sec.
- Incubate tubes on ice for 2min.
- Add 300μL LB to cells and incubate shaking at 37° C for 1hr.
- Spread cells on selective media
- Incubate plates overnight at 37° C.
Isolation of DNA for Colony PCR
GeneReleaser is a proprietary reagent that releases DNA from cells while sequestering cell lysis products that might inhibit DNA polymerases.- Resuspend the GeneReleaser through inversion, not vortexing. Add 20μL GeneReleaser to each PCR tube.
- Add cells from plates with a sterile pipette tip with 10μL of appropriate liquid media OR 10μL from overnight liquid culture.
- Run PCR tubes on following thermal cycle program:
- DNA will be in the clear liquid above the white precipitate at bottom of tube.
| Temperature (°C) | Time (sec) |
|---|---|
| 65 | 30 |
| 8 | 30 |
| 65 | 90 |
| 97 | 180 |
| 8 | 60 |
| 65 | 180 |
| 97 | 60 |
| 65 | 60 |
| 80 | hold |
Agarose Gel Electrophoresis
- To make a 0.7% agarose content gel first add 0.21g agarose and then 30mL 1 X TBE buffer to a 250mL Erlenmeyer flask.
- Microwave until the solution boils, about 45-60sec. Let boil for 5sec, then check for agarose that has not gone into solution. If there is undissolved agarose, boil for 5sec at a time until solution is homogeneous.
- Let solution sit until it is cool enough to touch and then add 2μL ethidium bromide using caution and swirl mixture.
- Set up gel tray and combs and pour gel until it is solidified, about 30min.
- Place gel in chamber oriented with positive electrode at the bottom of the gel and cover with 1X TBE.
- Add 5μL water, 5μL DNA, and 2μL 6X loading dye.
- Remove the comb and load each sample along with 10μL of a 1kb ladder. Run at 100 volts.
- When loading dye has run to the end of the gel, remove gel.
Colony PCR
- Prepare primers as followed
- Spin down at 13300 rpm for 50sec.
- Add appropriate amount of nuclease free water to make a 100μM stock solution, from which a 20μM working solution is made.
- Make the solution for PCR according to the following recipe
- The resulting mixture we got from DNA isolation (DNA in the clear liquid above and GeneReleaser at the bottom)
- 6.5μL nuclease free water
- 5μL buffer
- 0.5μL dNTP (10μM)
- 1μL left primer
- 1μL right primer
- 0.6μL DMSO
- 0.5μL DNA polymerase
- Run PCR tubes on following thermal cycle program
- Take amplified DNA from the clear liquid layer on the top.
| Step | Temperature (°C) | Time (sec) |
|---|---|---|
| 1 | 98 | 60 |
| 2 | 98 | 10 |
| 3 | 3°C above the Tm of the primer (without prefix/suffix) that has the lower Tm of the two | 30 |
| 4 | 72 | extention rate at 30 sec/kb |
| repeat step 2 to 4 for 5 times | ||
| 5 | 98 | 10 |
| 6 | 3°C above the Tm of the primer (with prefix/suffix) that has the lower Tm of the two | 30 |
| 7 | 72 | extention rate at 30 sec/kb |
| repeat step 5 to 7 for 25 times | ||
| 8 | 72 | 300 |
| 9 | 4 | hold |
| DNA sample | Temperature used in step 3(°C) | Temperature used in step 6(°C) | Time used for extention steps(°C) |
|---|---|---|---|
| rsaA | 60 | 72.1 | 30 |
| esp | 41.2 | 68.4 | 45 |
| rsaA Promotor | 61.6 | 73.4 | 30 |
| Pxyl | 49.2 | 71.4 | 30 |
DNA Purification by Centrifugation
- Make an SV Minicolumn assembly by placing a minicolumn in a collection tube.
- Transfer impure DNA solution to minicolumn assembly and incubate at rt for 1min.
- Centrifuge assembly for 1min at 16,000 x g (14krpm). Remove minicolumn from collection tube and discard liquid in collection tube. Reassemble assembly.
- Wash minicolumn by adding 700μL Membrane Wash Solution, previously diluted with 95% EtOH, to minicolumn and centrifuging as in step 3. Discard liquid in collection tube.
- Wash again with 500μL of wash solution, this time centrifuging for 5min at 16,000 x g.
- Discard liquid in collection tube. Centrifuge for 1min with microcentrifuge lid off or open to allow any remaining EtOH to evaporate.
- Transfer minicolumn to a clean 1.5mL microcentrifuge tube and add 50μL nuclease-free H2O to column membrane without touching the membrane with the pipette tip. Incubate at rt for 1min, then centrifuge as in step 3.
- Discard the minicolumn and chill the microcentrifuge tube that contains the eluted DNA.
