20.109(F12): Quick Facts

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(Homologous Recombination)
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==Homologous Recombination==
==Homologous Recombination==
Basic fact check: In the "chewback" step, we leave a 3' overhang. What is the direction that the enzyme that performs the "chewback" going in?
Basic fact check: In the "chewback" step, we leave a 3' overhang. What is the direction that the enzyme that performs the "chewback" going in?
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==Cloning==
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What are different ways after purifying whole plasmid to ensure that the plasmid is exactly correct?

Revision as of 11:34, 13 September 2012

Contents

DNA Polymerases

Taq Polymerase

  • Taq has an error rate of about 1 in 9000. Reference: an article in the journal Biochemistry, quick info can be found in the abstract.
    • So would you use Taq for cloning purposes?
    • What is the probability of finding a mutation in a GFP gene PCR-ed up using Taq polymerase?
    • If we start with 1 copy of GFP in a PCR reaction, then after 30 cycles, how many copies of GFP do we expect to have undergone one mutation (either away from WT, or reverting to WT)?
    • How many copies of GFP do we expect to have a mutation away from WT after 30 cycles, starting with 1 copy?
  • If you had a plasmid of size 3 kb, at a concentration typical of high copy plasmids after a mini prep (via Qiagen kits) (~150 ng/µL), and you added 1 µL of DNA into a PCR reaction (50 µL), given the typical conditions for a PCR, what are the concentrations of plasmid and primers respectively?
    • Would the concentrations be sufficient to explain why primers preferentially bind to the template compared to re-annealing of the template? If so, why? If not, why?

P.S. to all the 20.109 students, these are questions that have gone through my head, but I haven't written down an answer for. Lemme know if you've got an answer! *Eric Ma 16:47, 8 September 2012 (EDT):


Homologous Recombination

Basic fact check: In the "chewback" step, we leave a 3' overhang. What is the direction that the enzyme that performs the "chewback" going in?

Cloning

What are different ways after purifying whole plasmid to ensure that the plasmid is exactly correct?

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