March 4 Flashcards
(22 cards)
5’ to 3’ directional cloning is what direction (fwd/bwd), (-/+), bottom/top, and sense/anti-sense?
5’ to 3’ directional is forward, +, top, and sense
What is directional vs non-directional cloning?
Directional has insert going into vector in a direction. Non is when it can insert in either orientation
When you start using large plasmids (final plasmid size after inserting gene into vector), would you use low or high copy plasmid?
replication is problem, would use low-copy plasmid.
Low or high copy plasmid for protein expression?
Low copy
The gene insert/plasmid needs to have ______ start and _____ codon. What else must be in frame and what does not need to be?
in-frame start codon, and a stop codon. Everything must be in frame with any other tags or proteins. Transcription start/stop and poly A tails do not need to be in frame
How do you leave the vector and insert in the purified state? How modify them before insertion? Does buffer matter?
Both are left with blunt ends and are linear. Do double digestion, may be buffer dependent, so may need buffer compatible with both enzymes or do 2 independent digestions.
Are blunt ligations less or more efficient than sticky? What can help ligation efficiency?
They are less efficient. Macromolecules like polyethylene glycol.
When do ligation, do you want more of insert or vector?
Want more insert than vector.
How figure out how much insert DNA needed in ng based on vector ng and ligation ration?
Have vector ng x (insert length/vector length) athen multiply by the ratio of insert to vector. So vector cancels out, so does the lengths, so left with insert ng.
How can vector self-ligation be prevented?
Can use more insert than vector, and use phosphatase
What conditions does vector need after double digest to be able to self ligate?
It needs compatible ends and 5’ phosphates
How do you create digested vector so won’t self ligate? What need to do after ligation with insert?
You need to treat it with phosphtase, just leaves OH groups on all ends of all strands. After insert ligation, Still have 2 nicks as 5’ OH still there from vector. Still stable enough to go into bacteria, then endogenous enzymes repair it
What are the 3 ways that bacteria uptake DNA?
Conjugation, transduction, transformation.
What does heat-shock transformation do to bacteria?
Cells are incubated with CaCl2, so membranes more permeable. Then heat shock induces pores in MB so plasmid can enter
How transformation done with electroporation?
Have cell but no salt or anything. Use pulse of high voltage electicity to open membranes
What are units of transformation efficiency for bacteria? What is good range?
It is number of colonies per amount of DNA (CFU/ug). This uses the amount of starting DNA in vector form. Want 10^6 to 10^9 CFU/ug
How do blue-white screening to determine if colony has insert or not?`
No insert means that the lacZa gene will have B-gal that will hydrolyze X-gal to give blue. If inserted into MCS, disrupts lacZa expression so no B-gal, so gives white
What is colony PCR?
It is when you mix bacteria/yeast in PCR mix (lyse them), so their DNA is amplified. If plasmid present, will see it
How does insert-specific, backbone specifiic, and orientation specific primers affect the postive and negative clones?
Insert specific: No PCR product from neg clones, so no band on gel. Both primers anneal to insert DNA.
Backbone specific: Positive clone will have longer PCR product as the insert would have gone in, but don’t know what orientation insert is in.
Orientation specific primers: Negative clones will have no PCR product. It is when 1 primer anneals to insert, other to the backbone.
How is positive selection done to determine if the inserted vector also has the insert in it?
There is the ccdB gene in MCS, that is lethal when expressed. Insert ligation disrupts it, so cells with the plasmid that also have insert will survive. If vector self ligates and inserts, still get it expressed
How can you determine if the insert ligated into the plasmid if the insert is very small?
Do sequencing, this is expensive and time consuming, but can identify if plasmid has the extra nucleotides from the insert
