PCR purification and sequencing Flashcards
What does buffer PB do?
Binds the PCR product the single and double stranded, and washes away the primers
Why do we wash the product with salts and water?
To elute teh DNA and wash away the contaminants while the DNA will adsorb to the silica membrane
Why is important to clean up the PCR products to get rid if primers if you are going to set the set the sequencing reaction with the same primer?
Because we want a correct reading and not multiple ones, you won’t be able to correctly read your sequence but with primers you won’t know where your sequence is
Why was your purified PCR product been eluted with water and not TE?
So it doesn’t interfere with the products
What’s the difference in preparing a PCR and DNA sequencing?
DNA sequencing uses labelled ddNTP that doesn’‘t allow extension of the product and acts as a chain terminator
While PCR uses both dNTP and ddNTP
What else is included in the sanger sequencing?
1Primer to create a complimentary strand,
What’s the difference between ddNTP and dNTP molecularly?
ddNTP has a hydrogen group instead of hyroxyl
How is the sanger sequencing done?
4 reactions are done, they add each ddNTP in each tube with the primers, the template is synthesized until it encounters the ddNTP. The products are heat dentured and then run on gel and each lane has each reactio and it’s detected by autoradiography. Can also be done by capillary electrophoresis the ddNTP will be fluorescently labelled
How do you read the sequence on the gel?
You read from left to right, bottom to top
How can the length of the sequence change? How does it change
By changing the primer to template ratio
If it’s high then there will be stronger signal close to the primer but weaker ones further away
If it’s low it’s the opposite
ddNTP:dNTP
What is the big dye made of?
terminating sequence reagent, labelled binding dye and dNTP and primers
Why are a mixture of dNTP and ddnTP used and not just ddNTP?
Because it needs the primer to be different so it can be visualized and sequenced compared to the labelled termination of the sequnce
What’s the fundamental difference between PCR and sequencing amplification?
There is a cooling persiod in the PCR where the strands ligate and they’re double stranded but for sequencing there isn’t this step
