Team:Amsterdam/project/protocols/
From 2012.igem.org
(Difference between revisions)
Line 88: | Line 88: | ||
- | + | Notes:<ul> | |
<li>Keep the 100% ethanol at -20˚C. The ethanol must always be cold prior to use.</li> | <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> | + | <li>Centrifugation at 4˚C is preferable.</li></ul><br><br> |
+ | |||
+ | |||
+ | <h2>Preparation of Competent E. coli</h2> | ||
+ | |||
+ | <h3>Preparation of Reagents</h3> | ||
+ | |||
+ | <li>Buffer 1:<ul> | ||
+ | <li>30 mM KOAc, 100 mM RbC12, 10 mM CaCl2, 50 mM MnC12, 15% glycerol, pH 5.8</li> | ||
+ | <li>For 500 ml: 1.47 g KOAc (MW 98.14) </li> | ||
+ | <li>6.04 g RbC12 (MW 120.92) </li> | ||
+ | <li>0.74 g CaC12 (MW 147.02) </li> | ||
+ | <li>4.94 g MnC12-4H20 (MW 197.9) </li> | ||
+ | <li>75 ml glycerol </li> | ||
+ | <li>pH to 5.8 with dilute acetic acid.</li></u></li> | ||
+ | <li>Filter sterilize</li> | ||
+ | <li>Buffer 2:</ul> | ||
+ | <li>10 mM MOPS/KOH pH 6.5, 75 mM CaC12, 10 mM RbC12, 15% glycerol</li> | ||
+ | <li>For 100 ml: 0.21 g MOPS (MW 209.26) </li> | ||
+ | <li>1.10 g CaC12 (MW 147.02)</li> | ||
+ | <li>0.12 g RbC12 (MW 120.92) </li> | ||
+ | <li>15 ml glycerol </li> | ||
+ | <li>pH to 6.5 with 1 N KOH.</li></ul></li> | ||
+ | <li>Filter sterilize.</li><br><br> | ||
+ | |||
+ | <h3>Protocol</h3> | ||
+ | |||
+ | <li>Streak desired E. coli strain on fresh LB plate. Grow overnight at 37°C. </li> | ||
+ | <li>Inoculate a single colony into 5 ml LB. Grow overnight, shaking at 37° C. </li> | ||
+ | <li>Inoculate about 1 ml into 200 ml LB in a 2 1 flask. Shake at 37°C until OD550 = 0.5. </li> | ||
+ | <li>Chill flask in ice-water 5 minutes. </li> | ||
+ | <li>Spin 5 minutes at 6,000 rpm in GS3 rotor. </li> | ||
+ | <li>Resuspend pellet in 80 ml ice cold Buffer 1. </li> | ||
+ | <li>Chill in ice-water 5 minutes. </li> | ||
+ | <li>Spin 5 minutes at 6,000 rpm in GS3 rotor. </li> | ||
+ | <li>Resuspend pellet in 8 ml ice cold Buffer 2. </li> | ||
+ | <li>Chill in ice-water 15 minutes. </li> | ||
+ | <li>Make 50, 100 and 200 ml aliquots in 1.5 ml Eppendorf tubes.</li> | ||
+ | <li>Flash-freeze in liquid nitrogen. </li> | ||
+ | <li>Store at -80oC.</li><br><br> | ||
+ | |||
+ | |||
+ | Notes:<ul> | ||
+ | <li>Keep buffers, tips, tubes, rotors, etc. ice cold</li></ul><br><br> | ||
</p> | </p> | ||
</div> | </div> |
Revision as of 22:33, 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
Notes:
- Keep the 100% ethanol at -20˚C. The ethanol must always be cold prior to use.
- Centrifugation at 4˚C is preferable.
Preparation of Competent E. coli
Preparation of Reagents
- 30 mM KOAc, 100 mM RbC12, 10 mM CaCl2, 50 mM MnC12, 15% glycerol, pH 5.8
- For 500 ml: 1.47 g KOAc (MW 98.14)
- 6.04 g RbC12 (MW 120.92)
- 0.74 g CaC12 (MW 147.02)
- 4.94 g MnC12-4H20 (MW 197.9)
- 75 ml glycerol
- pH to 5.8 with dilute acetic acid.
Protocol
Notes:
- Keep buffers, tips, tubes, rotors, etc. ice cold