Plasmid Design Flashcards
When asked to explain the strategy of DNA insertion what should your answer include?
- Describe the treatment of the plasmid of gene with restriction enzymes and at which sites they’ll cut at (include diagram of plasmid having it’s DNA section removed + write down the specific sticky ends at each site of both the plasmid and insertion DNA if asked for the )
- Introduce them together and treat them with DNA ligase enzymes to form recombinant plasmid
Why are blunt ends harder to use
They have the tendency to ligate back together
Marker of Kinase site
can be tagged by P32 (radioactive)
Function of Histags
Can be used to purify the protein in a nickel column (downstream thrombin allow for histag cleavage post purification)
Steps of primer design:
When designing primers, you design a forward and reverse primer. The forward primer is the first 21 base of the sense strand (5’ to 3’), with an the restriction site and repetitive region added to the be 5’ end (left).
The reverse strand is more complex:
Step 1: write the target sequence (last 21 bases) 5’ to 3’, adding the restriction site onto the 3’ end and repetitive adenine region (the length is determined by the difference between the length of the 21 bases and restriction site and the requested length of the primers.)
Step 2: Write the complementary sequence (making it become 3’ to 5’) as it represents the antisense strand.
Step 3: to make it back to 5’ to 3’, to allow binding to the target antisense strand, reverse the sequence from right to left.
What is a primer:
A primer is a single stranded oligonucleotide of around 21 bp, 18 of which are complementary to a binding, 6 bp restriction enzyme site, 4-6 before the restriction enzyme site to ensure stable binding of the restriction enzyme.
Stages of PCR
Denaturation (separate DNA strands), Annealing (primer attaches), elongation (DNA replication)
