Team:Glasgow/Notebook/Protocols
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Programme Settings | Programme Settings | ||
+ | <ol> | ||
+ | <li>98⁰C – 1 min</li> | ||
+ | <li>98⁰C – 20 sec</li> | ||
+ | <li>55⁰C – 30 sec</li> | ||
+ | <li>55⁰C – 30 sec (Back to step 2. x30)</li> | ||
+ | <li>72⁰C – 10 min</li> | ||
+ | <li>4⁰C – Hold</li> | ||
+ | </ol> | ||
+ | |||
+ | |||
+ | |||
+ | <!-- | ||
<table> | <table> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
</table> | </table> | ||
+ | --> | ||
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<p id="RestrictionDigests"> | <p id="RestrictionDigests"> | ||
- | <h2></h2> | + | <h2>Restriction Digests</h2> |
<ol> | <ol> | ||
- | <li></li> | + | <li>Add |
- | <li></li> | + | |
- | <li></li> | + | a. For a 20μl sample |
- | <li></li> | + | <ol> |
- | <li></li> | + | <li>2μl 10 x buffer</li> |
- | <li></li> | + | <li>4μl DNA sample</li> |
- | <li></li> | + | <li>14μl ddH20</li> |
- | <li></li> | + | <li>0.5μl restriction enzyme</li> |
+ | </ol> | ||
+ | |||
+ | b. For a 30μl sample | ||
+ | <ol> | ||
+ | <li>3μl 10 x buffer</li> | ||
+ | <li>10μl DNA sample</li> | ||
+ | <li>17μl ddH20</li> | ||
+ | <li>0.75μl restriction enzyme</li> | ||
+ | </ol> | ||
+ | |||
+ | </li> | ||
+ | <li>Mix thoroughly</li> | ||
+ | <li>Incubate at 37⁰C for at least an hour</li> | ||
+ | |||
</ol> | </ol> | ||
Revision as of 15:12, 15 October 2014
Protocols
EditThis page shows our protocols for various lab procedures.
Creating Chemically Competent Cells
- Pipette 400μl of culture into 20ml of fresh broth
- Incubate the cultures in shaking 37⁰C water bath for 90 min
- Calcium chloride and centrifuge tubes must be cooled on ice.
- Pour culture into centrifuge tubes, return to ice.
- Centrifuge in a cold rotor for 2 min at 7000rpm (6000 G)
- Pour out the supernatant, being careful about the pellet
- Add 10μl of calcium chloride, immediately return to ice and leave for 1 hour (or longer)
- Repeat the centrifuge step, pour out supernatant
- Store on ice, add 1ml of calcium chloride. Resuspend and return to ice.
Transformation of Competent Cells
- Add 100μl of competent cells to cooled Eppendorfs
- Add 1μl of DNA
- Incubate on ice for 20 min
- Heat shock at 37⁰C for 5 min
- Place immediately on ice and leave for 5 min.
- Add 200μl of L-Broth and incubate at 37⁰C for 90 min. Expression step.
- Spread 100-200μl on full dried medias. On half plates use 40μl.
- Incubate plates
DNA Clean-up
- Transfer supernatant to a mini-column
- Spin for 1 min on full and pour out flowthrough
- Add 500μl of PB, spin for 1 min and discard flowthrough
- Add 800μl of PE, spin for 1 min and discard flowthrough
- Spin again and discard flowthrough.
- Add 50μl of EB to the centre of the white disc and stand for 1 min.
- Move column to a 1.5ml Eppendorf (leaving behind the collection tube) and spin for 1 min.
- Keep supernatant. DNA is in the liquid.
PCR
50μl reaction volume –- 10μl 5 x HF Buffer
- 5μl 5mM dNTPs
- 1μl 50mM MgCl2
- 1.5μl DMSO
- 5μl 5μM F primer
- 5μl 5μM R Primer
- 22μl ddH2O
- 1μl (1/100 dilution) template DNA
- 0.5μl polymerase
- 98⁰C – 1 min
- 98⁰C – 20 sec
- 55⁰C – 30 sec
- 55⁰C – 30 sec (Back to step 2. x30)
- 72⁰C – 10 min
- 4⁰C – Hold
Restriction Digests
- Add
a. For a 20μl sample
- 2μl 10 x buffer
- 4μl DNA sample
- 14μl ddH20
- 0.5μl restriction enzyme
- 3μl 10 x buffer
- 10μl DNA sample
- 17μl ddH20
- 0.75μl restriction enzyme
- Mix thoroughly
- Incubate at 37⁰C for at least an hour