Smolke:Protocols/Screening: Difference between revisions
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==Josh's Protocol== | ==Josh's Protocol== | ||
===Sample Preparation=== | ===Sample Preparation=== | ||
*Aliquot 50 uL water into sterile PCR tubes, one tube per colony. | *Aliquot 50 uL sterile water into sterile PCR tubes, one tube per colony. | ||
*Using a P20, scape off a colony and resuspend it in the water. | *Using a P20, scape off a colony and resuspend it in the water. | ||
*Use 1 uL of this cell suspension as your PCR template | *Use 1 uL of this cell suspension as your PCR template | ||
**Tubes can be stored at 4C for several days. Use 5 uL of cell suspension to inoculate a 5 mL overnight culture. | |||
===Reaction Mixture=== | ===Reaction Mixture=== | ||
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**0.2 uL 1:10 Taq | **0.2 uL 1:10 Taq | ||
**7.2 uL water | **7.2 uL water | ||
*Note: Make a master mix of everything except the template. Aliquot 9 uL into a separate set of PCR tubes. Then transfer 1 uL of cell suspension to each tube. You can use a multichannel pipet to transfer up to eight templates at once. | |||
***Run PCR as normal, | *Run PCR as normal, with the first step changed to a 7 minute incubation at 95C (5 minutes to lyse + 2 minutes to denature DNA). | ||
==Yvonne's Protocol== | |||
===Procedure=== | |||
*Warm up necessary number of LB (+ appropriate antibiotics) plates for restreak | |||
*Make master mix; include 1 or 2 extra volumes to account for errors from repetitive pipetting. Remember to account of positive and negative control samples. For each sample: | |||
**19.5 ul water | |||
**2.5 ul 10x Taq buffer | |||
**1.25 ul 50 mM MgCl2 | |||
**0.5 ul Fwd primer | |||
**0.5 ul Rev primer | |||
**0.5 ul dNTP | |||
*Line up necessary number of PCR tubes | |||
*Take out pre-warmed LB plate and label with colony numbers | |||
*Add 0.25 ul 1:10 Taq DNA polymerase for each sample to master mix; pipette or invert tube to ensure thorough mixing | |||
*Aliqout 25 ul of master mix to each tube | |||
*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 | |||
**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 | |||
*Load 15 ul of each PCR product on agarose gel for analysis | |||
===Notes=== | |||
*Ideally, use primer sets that would generate a band for both positive and negative colonies but with different sizes. | |||
*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. | |||
*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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