Team:Amsterdam/project/protocols/
From 2012.igem.org
(Difference between revisions)
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<li>The exonuclease amount is ideal for the assembly of DNA molecules with 20– 150 bp overlaps.</li> | <li>The exonuclease amount is ideal for the assembly of DNA molecules with 20– 150 bp overlaps.</li> | ||
<li>Between 10 and 100 ng of each 6 kb DNA fragment was added.</li> | <li>Between 10 and 100 ng of each 6 kb DNA fragment was added.</li> | ||
- | <li>For larger DNA segments, proportional amounts of DNA should be added (for example, 250 ng of each 150 kb DNA segment).</li> | + | <li>For larger DNA segments, proportional amounts of DNA should be added (for example, 250 ng of each 150 kb DNA segment).</li></ul><br><br> |
+ | |||
+ | <h2>DNA Precipitation</h2> | ||
+ | <li>Add 1/10 of sodium acetate (3M, pH 5.2) to the total volume of DNA to be precipitated. Vortex to make sure that the solution is well mixed.</li> | ||
+ | <li>Add 2.5 X total volume in eppendorf of 100% ethanol. Vortex and keep the mixture at -20˚C for 30 minutes.</li> | ||
+ | <li>Centrifuge at maximum speed for 20 minutes.</li> | ||
+ | <li>Discard the supernatant without disturbing the pellet and add about 500 µl of 70% ethanol.</li> | ||
+ | <li>Centrifuge for one minute at maximum speed. Discard the supernatant.</li> | ||
+ | <li>Add about 500 µl of 70% ethanol again. Centrifuge for one minute at maximum speed. Discard the supernatant.</li> | ||
+ | <li>Remove as much ethanol as possible using a glass Pasteur pipette. Allow to air dry.</li> | ||
+ | <li>Resuspend the DNA in 20 µl of deionised sterile water.</li><br><br> | ||
+ | |||
+ | |||
+ | <li>Notes:<ul> | ||
+ | <li>Keep the 100% ethanol at -20˚C. The ethanol must always be cold prior to use.</li> | ||
+ | <li>Centrifugation at 4˚C is preferable.</li> | ||
</p> | </p> | ||
</div> | </div> |
Revision as of 22:28, 31 July 2012
Protocols
Transformation Protocol in DH5α (Invitrogen)
Notes:
- Do not shake the polypropylene tubes during the heat shock!
- To increase the yield, centrifuge gently (at low speed), remove the excess of medium and then spread on the plates.
- To determine transformation efficiency
Gel Electrophoresis
- 5 µl DNA ladder
- 5 µl sample + 5 µl H2O + 1 µl loading buffer
Preparation of LB Medium and Agar Plates
- Bacto Tryptone – 10 grams
- NaCl – 10 grams
- Yeast extract – 5 grams
Gibson Assembly
Preparation of reagents
- 3 ml of 1M Tris-HCl pH 7.5
- 150 ml of 2M MgCl2
- 60 ml of 100mM dNTP
- 300 ml of 1M DTT
- 1,5 g PEG-8000
- 300 ml of 100mM NAD
One-step isothermal DNA assembly
protocol (Gibson Reaction)
- 8 µl 5X isothermal buffer
- 0.8 µl of 0.2 U.µl –1 or 1.0 U.µl –1 T5 exonuclease
- 4 µl of 40 U.µl –1 Taq DNA ligase
- 0.5 µl of 2 U.µl –1 Phusion DNA polymerase.
- 5 µl of DNA
- Water up to 40 µl
Notes:
- All isothermal assembly components can be stored at –20°C in a single mixture at 1.33X concentration for more than one year. The enzymes are still active after more than ten freeze-thaw cycles. The aliquots should be kept on ice until ready to use.
- The exonuclease amount is ideal for the assembly of DNA molecules with 20– 150 bp overlaps.
- Between 10 and 100 ng of each 6 kb DNA fragment was added.
- For larger DNA segments, proportional amounts of DNA should be added (for example, 250 ng of each 150 kb DNA segment).
DNA Precipitation
- Keep the 100% ethanol at -20˚C. The ethanol must always be cold prior to use.
- Centrifugation at 4˚C is preferable.