Smolke:Protocols/Screening: Difference between revisions
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**19.5 ul water | **19.5 ul water | ||
**2.5 ul 10x Taq buffer | **2.5 ul 10x Taq buffer | ||
**1.25 ul | **1.25 ul 50 mM MgCl2 | ||
**0.5 ul Fwd primer | **0.5 ul Fwd primer | ||
**0.5 ul Rev primer | **0.5 ul Rev primer | ||
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*Pick up each colony with a toothpick, dip into PCR tube (containing master mix) and swirl, then restreak on LB plate | *Pick up each colony with a toothpick, dip into PCR tube (containing master mix) and swirl, then restreak on LB plate | ||
*Run PCR as normal, except the first step is changed to 95C for 5 min for cell lysis | *Run PCR as normal, except the first step is changed to 95C for 5 min for cell lysis | ||
**Note: The second step is 94C for 2 min, and this step is repeated each cycle while the first lysis step is not | |||
*Put restreaked plate back in incubator; restreaked colonies should be ready for inoculating liquid cultures in 4 hours | *Put restreaked plate back in incubator; restreaked colonies should be ready for inoculating liquid cultures in 4 hours | ||
*Load 15 ul of each PCR product on agarose gel for analysis | *Load 15 ul of each PCR product on agarose gel for analysis | ||
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*Should always include a negative control using the parent vector, no DNA template, or another appropriate choice. This would let you know if you have primer dimer or other nonspecific amplification. | *Should always include a negative control using the parent vector, no DNA template, or another appropriate choice. This would let you know if you have primer dimer or other nonspecific amplification. | ||
*Should include a positive control if possible. This is especially important if your chosen primers would only generate a band for positive but not negative colonies. | *Should include a positive control if possible. This is especially important if your chosen primers would only generate a band for positive but not negative colonies. | ||
*I use toothpicks whenever possible. They are much cheaper and more environmentally friendly (I believe) than pipet tips. |
Latest revision as of 16:15, 26 September 2010
Josh's ProtocolSample Preparation
Reaction Mixture
Yvonne's ProtocolProcedure
Notes
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