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Bio. 208 - Dilution of exonuclease treated samples
Terms in this set (13)
Why was the initial negative control reaction carried through to nested PCR?
in order to dictate where contamination occurred and specifically in which reaction the contamination took place in.
Why wasn't the initial positive control pGAP carried through to nested PCR?
Positive controls are used to ensure that the experiment was running correctly. For this reason, there should be no contamination in the positive control.
Why were both Jacaranda initial PCR reactions carried through to nested PCR?
Both Jacaranda initial PCR reactions were carried through to nested PCR in the event that a sample of DNA was damaged or contaminated, the other sample could always be used.
Why was a new negative control reaction made for nested PCR?
helps you dictate where contamination occurred, degenerate or nested PCR
How was the mastermix for this round of PCR different from the mastermix for the initial PCR?
the yellow primers are more specific for the new mastermix
Why was a 1/50 dilution made for the Jacaranda initial PCR samples as well as the continued negative control and Arabidopsis gDNA prior to adding to the mastermix?
It helps the unwanted products go away with the solution and encourages the primers for nested PCR to find the right product (should be in high abundance than non specific bases) to bind to the specific set of DNA
Why wasn't it necessary to dilute the new negative control or new pGAP positive control?
because the new negative control was just sterile water, so there was no need to dilute water with water. It wasn't necessary to dilute the new plasmid control because the plasmid control is just there to make sure that the machinery for PCR was working, and it also had not been amplified in any way because it was new and had not gone through degenerate PCR
What were the thermocycler conditions for the nested PCR reactions, and how did these conditions differ from the initial PCR?
1. Denaturation took place at 95 degrees celsius
2. Annealing took place at 46 degrees celsius
3. Extension took place at 72 degrees celsius
Why does the nested PCR reaction have a lower annealing temperature compared to the degenerate PCR?
The nested PCR reaction has a lower annealing temperature because we want there to be as much binding as possible as a result of there not having a lot of nonspecific regions in nested. The degenerate PCR has a higher annealing temperature because it makes it harder for the primers to bind. The degenerate PCR was set at a higher temperature in order to avoid the PCR binding to non-specific areas.
What was the final dilution of the initial PCR sample when it was in the second PCR tube mixed with mastermix?
the final dilution was 1 /50 um. this results from diluting 1% of the oringal sample
Why couldn't you just pipet 0.4 ul of the initial PCR sample in a final volume of 40 ul to make your dilution?
There would be an imbalance of the master mix and dilution in which there would be far to much of the master mix for the solution.
Easier for us to pipette
Why were the PCR tubes kept on ice until all of the students were ready to run the thermocycler?
So the primers didn't bind to the template and DNA polymerase wouldn't polymerize the DNA strand
If a forward primer sequence is 5'GATCAGGCTATGCTA3' and the reverse primer sequence is 5'AGCTTAGCACGATAC3' what would you use for the annealing temperature for this PCR reaction? Show your work.
- Tm= 2(A+T) +4(G+C)
- Forward primer: Tm= 2(8)+4(7)=44 degrees
- Reverse primer: Tm=2(8)+4(7)= 44 degrees
- If they were different take the average of the two - primer sequences
- Subtract 5 degrees to get the annealing - -temperature
- Ta= 44-5=39 degrees
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