A Cloning Problem
Part A: Using this information, what must have happened during the cloning process?
In the normal situation, there will be a sticky end and a blunt end, so the vector will not be self ligate. But in this one, two ends are both sticky.
The LacZ gene will be self ligated and reform a vector. So it will still be blue.
Part B: If you isolate the plasmids from the blue bacterial colonies and sequence them, what do you predict will be the base sequence of the top DNA strand at the ligation site?
PCR and DNA Sequencing
The main priciple of PCR
Combinig Cloning with PCR
Use restriction enzyme to cut target piece off the strands
Clone from mRNA
Use reverse transcriptase to make cDNA from mRNA.
Real-time PCR(Quantitative PCR, Q-PCR)
- Measures amount of product after each cycle.
- Product produces a fluorescen signal
CT: cycle threshold.
We can use it to quantify how much in our original sample.
D.8 Because in every cycle, the amount of genes will double.
Different Q-PCR Techonologies
SYBR-green works really well when we just have a very specific single product.
Inexpensive but binds to all dsDNA. Only works if product is specific.
Expensive, but very specifc.
First Generation(Sanger) DNA Sequencing: relies on dideoxynucleotide terminators
Conceptually similar to endpoint PCR.
- Requires four reactions, a large gel and radioactivity.
- Gives only 100-200 base pairs per sequencing run.
DNA Sequencing Problem
7 As and plus the whole one.
dNTPs: dATP, dCTP, dGTP, dTTP.
3 = 2 + 1