20.109(F12): Quick Facts
From OpenWetWare
(Difference between revisions)
(→Taq Polymerase) |
Current revision (11:50, 13 September 2012) (view source) (→Cloning) |
||
| (5 intermediate revisions not shown.) | |||
| Line 4: | Line 4: | ||
** So would you use Taq for cloning purposes? | ** 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? | ** 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 (~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? | + | * 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? | + | ** 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! *'''[[User:Eric Ma|Eric Ma]] 16:47, 8 September 2012 (EDT)''': | 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! *'''[[User:Eric Ma|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? | ||
Current revision
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?
