Experiment 3 >> New Cell Line Part 2

P0G44 Expression Plasmid contains Flp Recombinase

AP258

PcDA5/FRT/TO Expression vector containing FRT site and GOI

AP257

Why were the primers chosen?

They matched the backbone and the gene of interest. Also AP450 is flag tagged so the primers make sure only the TELO2 portion is amplified

Why was the restriction digest performed with ECORV and NOT1?

To decrease the noise from amplification and purify the product from the gel

The 12 main steps of creating the new TREX cell line

1. Making needed plasmids from glycerol stocks

2. Plasmid Prep from LB broth

3. Nanodrop plasmid DNA to obtain concentration

4. PCR to amplify plasmid DNA

5. Gel Extraction

6. Restriction Digest

7. Ligation

8. Transformation

9. Plate on Agar

10. Plate 1 - 4 colonies in LB

11. Nandodrop Plasmid Prep

12. Send preps to sequencer to confirm ligation and transformation worked

What is “the insert”?

TELO2 plasmid AP450

What is “the backbone”?

The structure that carries the GOI and replicates it in bacteria (AP257)

Explain diagnostic restriction digest

Use to confirm the identity of a DNA molecule or recombinant plasmid

• The PCR product is cut with specific restriction enzymes ad analyzed on an agarose gel

• To verify cloning experiment succeeded

What a single diagnostic restriction digest?

Confirms total plasmid size

What’s a double restriction digest?

Verifies insertion orientation or internal fragments

Diagnostic restriction digest main steps

1. Use plasmid map (snapgene) to select compatible enzymes

2. Incubate DNA from PCR with restriction enzymes, buffer, and water

3. Run digested DNA into gel and run gel

4. Analyze bands under UV light, compare to predicted map

What kind of digest did I do?

A double digest

• two flanking enzymes were used

• 2 bands resulted, larger band is backbone smaller band is insert

Key PCR Ingredients

• Buffer

• dNTPs

• Primers

• Water

• Polymerase

What’s the buffer for in PCR?

Maintains stable pH and provides optimal chemical environment for DNA polymerase to function

What kind of cloning is the Flp-In TREX system?

Site specific recombinase mediated cloning

• FLP/FRT recombination

Using restriction enzymes and ligases is what type of cloning?

Golden Gate

What type of cloning uses exonuclease drive homologous overlaps?

Gibson Assembly

What makes Flp-In TREX so much different than golden gate or gibson assembly?

The system relies on the Flp recombinase enzyme

Why isn’t the RD and ligation step I did considered?

Because I didn’t use a Type IIS restriction enzyme (Bsal, BsmBI)

What types of enzymes are ECORV and NOT1?

Type II

not type IIS

What’s the biggest workflow difference between traditional restriction cloning and golden gate?

Traditional everything is done in steps: Digest DNA > purify fragments > ligate

Golden gate everything is done at once: digestion and ligation happen at the same time during thermal cycling, Type IIS enzymes cut outside their recognition sites

What is the purpose of the dNTPs?

building blocks for DNA polymerase to add onto growing DNA chain

What’s the purpose of the forward and reverse primers?

Starting points for DNA polymerase, added on either in of the target region to copy

Where does the forward primer bind?

At the start of the target sequence on the bottom antisense strand

Where does the reverse primer bind?

At the top (coding sense) starts replication in the reverse direction back toward the start

What was the purpose of water in PCR?

Reaction volume balancer

Purpose of Hifi Polymerase

High fidelity - will replicate dna with extreme accuracy and very low error rate

What makes Hifi Poly different from Taq?

A 3 to 5’ exonuclease proofreading domain

• has error correction will cut out the incorrect nucleotide immediately and replace it before starting again

How does hifi know if the incorrect nucleotide has been added?

• Nucleotides that match will fit into polymerases catalytic pocket

• If they don’t they ruin the shape of the DNA and the final shape will be a small bump that sticks out

• Replication will pause here because no nucleotide can attach onto misformed DNA, the open end detaches from the polymerization zone and falls into the proofreading domain…mismatched nucleotides are chopped off, then replication can begin again

What is EM-7 in the Tet repressor expression plasmid?

A bacterial promoter - functions to drive the expression of the selection marker gene, the blasticidin resistance gene

What’s the purpose of having two different bacterial promoters in the expression plasmid?

SV40 - for blasticidin resistance in mammalian cells during cell line creation

EM-7 - for blasticidin resistance in bacterial cells (e.coli) during plasmid prep