DNA cloning

Question 1

What are the 5 main steps of DNA cloning?

Answer 1

DNA digestion
Ligation with DNA ligase
Transformation
Selection
Recovery of recombinant plasmid

Question 2

Dam methylation blocks
DCM methylation blocks
Mcr system

Answer 2

MboI and Xbal
EcoRI
cuts DNA methylated A/GC

Question 3

Formula of frequency of restriction site cutting

Answer 3

F= 1 per 4^N where N= lenght of recognition site

Question 4

Ligation conditions

Answer 4

chance of vector finding
ratio vector/insert
size of DNA
concentration of DNA

Question 5

Alkaline Phosphatase function and how

Answer 5

prevents religation of the vector by transforming both 5´P ends by 5´OH (desphosphorilation)

Question 6

Where does DNA ligase come from and what does it do (requires and what direction)?

Answer 6

T4 phage
joins vector to plasmid by sealing 5´-3´using ATP

Question 7

Introduction of DNA can be done by two methods? How?

Answer 7

Transformation (chemical): permeabilization with Ca2+ - heat shock (0-42ºC) - selective medium
Electroporation (electric shock) - electrical discharge

Question 8

Selection can be done by two main techinques

Answer 8

physical (hybridization and PCR)
biological (phenotype and protein product)

Question 9

Blue-white screening

Answer 9

insertional inactivation of lacZ gene - B-gal not produced - X-gal not cleave - no blue colour released (white)

Question 10

Recovery of plasmid DNA three main steps

Answer 10

Production of a cleared lysate (denature - salt precipitation - spin- plasmids in supernatant)
Affinity purification (anion exchange or silica/glass particles)
Dye bouyant equilibrium density centrifugation (CsCl density gradient containing Ethidium Bromide - plasmid DNA denser lower in gradient)

Question 11

Three methods of PCR cloning vector and explain

Answer 11

Restriction sites in primers: at 5´ends of primers, add 2-6nt (efficiency), digest plasmid with compatible RE

TA cloning vector (Promega): PCR products have untemplated A on 3´ends and vector has unpaired T on
3´, Taq polymerase, blue-white screening

TOPO cloning vector (Thermo): vector w/ topoisomerase I at ends, PCR product with A overhangs, blunt end, 95% efficiency (blue-white and lethal ccdB gene with TOPO in middle to survival)

Question 12

explain Gibson Assembly (3 steps)

Answer 12

Overlapping PCR products (different inserts in same vector)

linearize vector: RE or inverse PCR (fragment fill gaps between primers)

PCR amplification: amplify insert 3´, vector specific tail 5´(anneal with ends of vector)

Gibson reaction: T5 exonuclease (chews 5´ends to overlap fragements), DNA polymerase (fills overhangs) and DNA ligase (seals nicks)

Question 13

Vectors features

Answer 13

maintenance (replicon) - free replication (ORI) and control copy number (rop)

RE site - unique and multiple
introduction into host - transformation or infection

selection marker - low transformants, Ab resistance

identify recombinants- easy screen and insertional inactivation

Question 14

Genomic Library

Answer 14

isolate chromosomal DNA
digest with RE
ligate chromosomal DNA with vector

Question 15

Formula of number of clones needed to find a fragment in a library

Answer 15

N= log e (1-0,95) / log e (1-(size cloned DNA/total genome)