6 Cloning

Question 1

What is cloning

What is it used for

Answer 1

A method used to make hundreds of copies of a DNA sequence, sues a plasmid usually called a vector for bacterial expression

This is so you can use the DNA directly for sequencing or as a substep in a larger cloning strategy (subcloning)

Or so you can over express the dna for RNA isolation , protien isolation (with or without having an epitope tag)

Transgenics (for in vivo)

Question 2

What are to two main components in cloning

What are they for

Answer 2

Vector and insert

Vector: The destination for the Gene you want to clone

Insert: gene you want to clone

Question 3

What are the general cloning steps

Answer 3

Vector prep

Insert prep

Ligation

Transformation

Colony screening

Question 4

What is directional and non directional cloning

Answer 4

Directional: the insert goes into vector in one specific direction
- 5-3 is forward and the top is the sense strand
- 3-5 is reverse and the bottom is antisense strand

Non directional: allows insert to go into vector in any direction (easier)

Question 5

What should be considered about the final plasmid size in cloning

Answer 5

If the plasmid is more than 20kb:

• replication becomes and problem (because need to use more resources to replicated) so you need to use a low copy plasmid (has a low copy origin of replication that doesn’t replicate many copies of the plasmid in the cell)
• transformation efficiency becomes a problem so use electroporation instead of heat shock to transform

Question 6

How did you know which ori to use in cloning

Answer 6

Low copy vs high copy

You want low copy for protien expression (because that protien could kill cells so to avoid lots of death just make less of that protien)

Also need to make sure if adding two plasmids into one cell that the plasmids are not incompatible with each other

Question 7

What is important for the insert if the goal is to express RNA or protien?

What if your making a transgenic

Answer 7

The insert goes downstream of the promoter in the plasmid and in the correct orientation (so correct strand is made)

The frame is the most important to consider , need to make sure insert is in frame with promoter , start and stop codon
- or tag if tag is downstream of insert
- also if making a transgenic where two genes are made need to make sure both in frame

Question 8

Overall Whag does the final protien from cloning need to have

Answer 8

Needs an in frame start and stop codon (either from the insert or the plasmid)

Needs to be in frame with any tags or other desired protiens

Question 9

What doesn’t need to be in frame with the insert

Answer 9

The transcription start/stop

Poly A signals

Question 10

What are the steps in vector preparation

Answer 10

1. RE digestion to linearize
   - treat with phosphatase after digest bc ends can self ligate
   - blunting of sticky ends from RE if need blunt instead
   - blunting via T-tailing (or other blunt to sticky reactions)
2. Or have already linearized vector via TOPO cloning

Then purify the digested vector

Question 11

What are the steps in insert isolation

Answer 11

Diff ways to get insert:

1. Plasmid digestion via RE to get the target gene if in the plasmid
   - then need to do blunting if want blunt ends
2. PCR
   - might need kinase because primer has not 5’ phosphate
   - might need blunting bc of 3’ A overhang made by Taq
3. Oligonucleotides
   - something that’s just short oligonucletides or get a bunch and overlap them then insert it

Purification HAS to happen after this

Question 12

What to consider for inserts in cloning

Answer 12

The vector and insert need compatible ands

They also need 5’ phosphates

The ligase can ligate any blunt peice, PCR form taq with A overhang to vector would have T overhang, Kd from RE digest sticky ends need to be compatible

Question 13

What to consider for double digestions in cloning

Answer 13

The restriction enzymes work best in certain buffer conditions

So you need to do a double digestion in a compatible buffer for BOTH RE

If not compatible buffer with one of the RE, star activity can happen where cut where it shouldn’t

This is also true for phosphatase,kinases, and blunting enzymes

or you could do two independent digestions BUT NEED TO PURIFY IN BETWEEN EACH

Question 14

Cloning requires multiple ___

Answer 14

Purifications, and anytime you do purification you lose DNA, at end you lose at least half the DNA

Digest-purify-dephos-purify

This is why it’s Important for the ligation to have high concentration of insert

Question 15

What to consider for ligation in cloning

What improves ligation efficiency

Answer 15

T4 dna ligase needs 5’ phosphate and 3’ OH

Compatible ends also required

Also blunt ligation are less efficient than sticky because ligase work better for sticky

Addition of inert macromolecules like PEG can improve ligation efficiency

Question 16

What is the ratio of insert to vector in ligation

Answer 16

More insert than vector to increase likelyhood of insert and vector binding

3:1 insert:vector

Slode 18 equation

Question 17

What can cause false positives in ligation

How to fix

Answer 17

Vector self ligation, this is because it passes the antibiotic screening
- insert self ligation will not pass the antibiotic screening so no false positives

Fix:

• prevented by using a phosphatase
• impair by using high ratio in insert to vector
• accounted for during screening by using a vector only control to see how eel the vector can ligate with itself

Question 18

When will a vector self ligate

How does phospahtase fix it

Answer 18

If it has compatible ends and at least one pair of 5’ phosphates (whether that comes from the insert or the vector)

Cause then nicks can be repaired later

Question 19

How does phospahtase fix vector self ligation but what is the problem

What else can this problem happen with

Answer 19

The phosphatase treated vector won’t self ligate but after the insert ligation two nicks are left (because no 5’ phos in vector to attach to insert)

But the vector with two nicks can still be taken up into bacterial cells and is repaired by endogenous enzymes

Nicks also happen if RE digested vector and blunt PCR insert
- the vector and still self ligate here too
- if the insert ligates there are still nicks

Question 20

Is insert self ligation a big problem

What is a problem

Answer 20

Not usually: if it does happen the circular ligated insert don’t have an ori or selection markers won’t do anything in The cell

But insert concatenation is a problem (two inserts with with compatible sticky ends come together) to instead of one insert you get two
- mainly happens if insert is very small or too much insert or ligase is used

Question 21

What is the optimal temp for ligation

Answer 21

14 - 25 deg for 10min to overnight

Higher temps means less times but also less yield

Question 22

What happens after ligation

Answer 22

Chemical transformation of the plasmid, don’t have to purify before hand Can

If doing electroporation transformation, need to purify before hand

Can also freeze after ligation

Question 23

What is the ligation control

Answer 23

Control where you just put in the vector however you treated it before the ligation and do ligation reaction with no insert)

This tells you how likely the vector is to actually self ligate

Also checks efficiency of phosphatase treatment of vector

Question 24

In transformation what are the three ways for bacteria to take up the dna

Answer 24

Conjugation

Transduction

Transformation (standard way we do it)

Question 25

What does it mean to make cells competent before taking up the plasmid dna

Answer 25

The cell membranes are made permeable So that the cells are able to absorb forgien DNA (the plasmid)

Question 26

What types of competent cells are there

Answer 26

Usually use E. coli cells Can makes cell chemically competent to do heat shock transformation or make cells electrocompetent to do electroporation transformations

Question 27

How do you know what strain to use for transformation

Answer 27

Type of strain depends on what you want to do later on like: Blue white screening Want to induce RNA expression If don’t or do want methylation use dam+/dcm- (cells that can or can’t methylate dna)

Question 28

What is heat shock transformation

Answer 28

Done on chemically competent cells Cells are incubated with CaCl2 to make their membranes more permeable Cheap fast and easy

Question 29

What is electroporation transformation

Answer 29

Done on electrocompetent cells A pulse of high voltage electricity opens the cell membranes Works better than heat shock for larger plasmids but needs specialized equipment Problem: Have to be more careful of salts in the solution cells are in bc using electricity If salt, ions cause greater voltage and electrical discharge (arcing), more damage to cells

Question 30

What is selection

Answer 30

Once plasmids in cells , selected to see if plasmid was taken up Uses the selectable marker (antibiotic resistance) in the vector Positive selection selects for the cells we want because presence of the plasmid allows the cell to live in the antibiotic Cells are grown on agar plates

Question 31

Whag is transformation efficiency How to calculates

Answer 31

The number of colonies made per amount of DNA ( units are CFU/microgram) Total number of colonies/microgram of starting amount of dna - if only a part of the transformation is plated and counted adjust the dna concentration accordingly Good competent cells range from 10^6-10^9 CFU/microgram

Question 32

What is the goal when getting colonies in transformations How can you see how successful the ligation was

Answer 32

Goal is to get single colonies (not touching) so we spread the solution on multiple plates To see how successful ligation was, compare the cut vector + ligase (control) to the cut vector + insert + ligase So if 10 colonies on vector only and 30 on vector + insert , this means 10 of the colonies in the vector + insert self ligated 1 in 3 self ligated

Question 33

What are the types of screening

Answer 33

Blue white Colony PCR RE mapping Postive selection (TOPO) sequencing

Question 34

What is blue white screening

Answer 34

Need bacterial cells with the native beta gal disrupted in their plasmids Then they’ll make the del15 (omega fragment of beta gal) Then the plasmid they take up need to have the beta gal alpha fragment Then put the cell with both your plasmid and its own on IPTG and X-gal medium If DNA inserted into MCS of your plasmid: white colony - the inserted DNA into the MCS disrupts lacZalpha expression - cell don’t have functional beta gal - X-gal in the media is not hydrolyzed - white colony If DNA not inserted into MCS of your plasmid: blue colony - plasmid without insert still has lacZalpha - so cell do have functional beta gal - X-gal in the media is hydrolyzed - blue colony

Question 35

What is colony PCR

Answer 35

Used to screen colonies from a transformation for the presence of insert Mix colonies of bacteria directly in the PCR reaction mix and do PCR with specific primers (the 95 degrees is enough to destroy the cells) Also save the Bactria use used for the screen (so you can have them is theyre postive for insert)

Question 36

Whag types of primers are used in colony PCR and Whag are the results and downsides

Answer 36

1. Inserts specific primer: - using primers that just amplify the insert - this means that negative clones (no insert, vector self ligated) will have no PCR products - no band on agarose Downside: - cant tell if there is actually no insert or if PCR just didn’t work 2. backbone-specific primer: - primer the do the backbone flanking the insert - a positive clone will have a longer PCR product than a negative clone (since postive is insert + backbone and negative is just backbone) - so this time the negative clone still has a PCR product Downside: - this doesn’t tell you the orientation of the insert since it just the back bone being primed 3. Orientation specific primers: - a primer on the insert and a primer in the back bone - will confirm the correct orientation of insert (bc primer on insert at one end and backbone on the other) - negative clones have no PCR product (bc one primer didn’t bind to the insert since no insert) - also if insert backward the primers will face the same way and no PCR product

Question 37

In what situation do you use back bone specific and orientation specific primers

Answer 37

If orientation is a concern (ex. want to do non directional cloning specifically) use both backbone and orientation specific primers (so have a control for negative colony If don’t care about direction the insert went in, do backbone specific primers

Question 38

Three primer PCR colony screening

Answer 38

That’s when you use one reverse primer as the backbone Primer Two forward primers: one for insert and one for backbone Now negative product gives the backbone bands and positive product gives backbone bands and orientation specific bands

Question 39

What is RE mapping

Answer 39

Another way to screen for inserted vector Make a plasmid map for the vector + insert Chose RE that will distinguish between the insert and plasmid - can do a cut site in vector and one in insert, or cut sites flanking the insert - there has to be more than one cut site in the plasmid (so you can see multiple bands) - so you do double digestions and make sure you can distinguish the product sizes on gel

Question 40

Explain positive selection cloning

Answer 40

But don’t confuse this with the positive selection to check if transformation of cells happens (using antibiotic) Mainly do if using TOPO cloning (from invitrogen) - This type of cloning uses the lethal ccdB gene in the MCS - the lethal gene is a negative selectable marker When an insert is ligated into the MCS, the ccdB gene is disrupted, cells with the correct vector+insert survive (postive selections) Cells with self ligated plasmids have intact lethal ccdB gene and die (negative selection), no colonies

Question 41

What is the sequencing screening

Answer 41

Just seqeunce the every colony that grew and check for insert sequence Expensive and time consuming But good if the desired plasmid is only different from the vector by only a few nucleotides (like if only trying to make a new plasmid with 1bp diff) - especially good if you first do other screens then use those colonies to seqeunce (so not sequencing so many colonies)

Question 42

Even if not using it as a screening step what is the final step of every ligation

Answer 42

Sequencing to make sure the ligation worked as intended Do this by sequencing at least the junctions between the vector and insert and the insert