7 Cloning Flashcards

Question

What is the sequence TOPO I recongnizes

Answer 1

They give the linearized vector linked to TOPO with: - either 3’ T overhangs (give insert with 3’ A overhang) - no overhangs (blunt), give blunt insert - or one end with 5’ GTGG overhang, give insert with compliment overhang First two are non directional Last is directional

Answer 2

Use disruption of the ccdB gene for positive selection Is insert not in, cells die

Answer 3

No because selecting for cells what have disrupted ccdB (they srivive) If self ligate the cells just die

Answer 4

The vector bound to TOPO Choose right kit for the PCR product you have : - Taq for TOPO-TA cloning - proofreading Taq for TOPO blunt cloning 5’ OH is needed so DONT kinase treat the insert Then topo ligates vector to insert Use ccdB to select for vector with insert Directional or non direction depending on type of TOPO cloning 30sec to 30 min reaction time

Answer 5

Does not use RE or ligases Uses the site specific recombination pathways of the lambda bacteriophage The attachment sequences of the phage DNA (attP) recombine with the attachment sequences of the bacterial chromosome (attB) This forms two new sites attL (left) and attR (right) So four diff types of dna sequences

Answer 6

Lambda phage: when phage and bacterial chromsomes merge: - attB + attP combine to make attL and attR when phage and bacterial chromsomes split : - attL + attR recombine out to make attB and attP In gateway: Both the vector and the insert have two att sites (ex. attP in vector and attB in insert), then there the BP and LR reactions: BP: attB from insert + attP from vector combine to make attL and attR LR: attL + attR combine to make attB and attP

Answer 7

1. The donor vector has 2 P sites flanking the ccdB gene and point to it: - these sites are P1 and P2 2. The PCR insert has 2 B sites flanking it and pointing to it: - these sites are B1 and B2 - B1 can only react with p1 and b2 with p2: - The sites need to be same orientation, number, and B with P or R with L. 3. When the two recombine, they make L1 and L2 sites flanking the insert in the vector. The previous ccdB gene from the vector recombines out to have the R1 and R2 sites Selection: any cells without the insert in the vector still have the ccdB gene plasmid and die Can clone different pieces together at the same time, to get a bunch of merged fragments B reacting with P is BP reaction, L reacting with R is the LR reaction, either one works just depending on what the company gives you

Answer 8

Ways to Create an entry clone: 1. PCR product with attB primers then combine with donor vector with attP: BP reaction (MOST COMMON) 2. TOPO clone the dna fragment into an entry vector: don’t need b and P sites , the TOPO inserts it 3. Subcloning : digest the DNA from the vector

Answer 9

Used to actually do further things with cloned fragment like: - protien expression in E. coli yeast insect and mammalian cells - protien tags - RNAi - reporter assays

Answer 10

There are single base pair mutations in both attB and attP which forces these mutations to pair with each other and nothing else

Answer 11

I think: Whag to use the B and P sites to connect multiple gene together

Answer 12

Need to kit which has entry vectors and desitination vector and all the enzyme to do the reaction Create insert using PCR: - need the att sites pointed toward each other and needs a pDONR for BP rxn - or proofreading taq (to make blunt PCR insert) and pENTR/TOPO (entry vector with P sites and for the TOPO reaction Or subcloning your insert from an entry vector (has your insert with L) to a destination vector with R (LR reaction) Directional: all P1 and P2 will be diff ends 1 hr reaction time, no phosphatase or kinase needed, ccdB helps with positive selection

Answer 13

Need overlapping sequences between two pieces of dna (vector and insert) Is one or two steps, isothermal, single tube reaction that can assemble up to 15 pieces together ASSEBMLES BIG PEICES so it’s good for dna that’s too big to be amplified by a single PCR (do two diff PCR and enough overlap with both PCR products to do Gibson reaction) Also good when you can’t find proper RE to digest and ligate that way

Answer 14

5’ exonuclease (cut 5’ ends only) DNA pol (fills in the gaps) DNA ligase (seals the nicks) YOU need to make own vector and insert and make sure they have overlaps which can ligate together All in one tube at one temp for one hr

Answer 15

Just has a higher fidelity polymerase You still have to give your own vector and inserts

Answer 16

1. Need vector and insert (or multiple insert to ligate together) with 15-30 bp overlaps 2. Insert is made via PCR and the primers are 30-60 bp long (because have overlap and your insert seqeunce they bind to) 3. Vector prep: - doesn’t need to be digested, can make primers on vector and do PCR to linearize/prepare it - if use RE, the reaction will blunt 5’ overhangs and fill in 3’ overhangs 4. Incubate vector and inserts with overlaps together, exonuclease cuts back 5’ ends to make sticky ends, they anneal, dna pol fills the gaps, ligase seals the nicks

Answer 17

Kit bought from NEB Need to get your own vector and insert (15-30 bp overlapping) If overlaps are present no extra steps needed DIRECTIONAL doesn’t need selection to check for correct assembly but does need screening 1 hr long

7 Cloning Flashcards

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