Oh yeah, totally possible.

You know how you get the DNA sequence your plasmid from a fasta file. I usually call that strand that you see in the fasta file the "+" strand. And the reverse complement (that you don't see in the fasta file) is the "-" strand.

The trick to do this with PCR is to select any window of nucleotides to work with.

Have the forward primer match several the "+" strand for several bases and go "clockwise". Then design the reverse primer to match the "-" strand for several bases. It is important that the reverse primer faces away from the forward-primer such that the reverse primer goes "counter clockwise" away from the forward primer.

The forward primer will use the minus strand as a template will amplify around the plasmid and run into a nick just before itself. The reverse primer will dock onto the the last residue that the forward primer amplified and the reverse primer will go around the product of the Forward primer reaction and run into a nick (because it has no complementarity to continue). The forward and reverse primers have no complementarity so they do not re-circularize the plasmid.

Primers are usually written: 5' -> 3'. The forward primer goes 5' -> 3' clockwise, the reverse primer goes 5' -> 3' but counter-clockwise.

It is important that the primers "face away" from each other. It is important that the 5' tails of the primers do NOT overlap at all. If the primer tails do overlap you will re-circularize the plasmid. Don't leave a base in-between the primers or you will miss a base, but do not overlap the tails of primers.

I guess it is possible that the primers could complete a full circle, but as long as the tails of the primers do not overlap then that shouldn't happen.

circular DNA of the same size runs a little different than linear DNA of the same size on an agarose gel. This should be about as easy as PCR amplifying an insert, and definitely easier than QuickChange mutagenesis because the primers are not designed to overlap. Since you are making linear DNA by the primers running into dead-ends where one starts the other ends, this amplification should actually be exponential.

I could draw you a picture explaining everything, but be warned that I am not an artist.

If you don't want to clip one base on a gene in your linearized DNA product, put the primers far from your gene. This is like a problem solving method without scissors.

If I have a circle made out of connected points and cut it anywhere I get a linear rope. I can also make a cut in a circular rope by drawing around the circle and ignoring the connection between two close points. That is what you are doing here.

This kind of "cut" is "blunt end". No bases are hanging over on any strand.

If you wanted to insert something into a plasmid you could do restriction enzyme digestion and ligation or you could do Gibson Assembly, just got to have the primers of the vector run away from the vector and overlap with the appropriate primers of the insert.

It is that easy in principle! Not as complicated as so many articles make an easy problem difficult.

Dear Trang

as Adron suggest you can just perform a vector PCR using an high fidelity Taq polimerase and long elongation type.

IT is standard approach to perform single point mutagenesis or to produce linearized plasmid suitable for enzime free cloning approaches as

- PIPE cloning (you can find some video about it on my blod: ProteoCool ( https://proteocool.blogspot.com/) at PAGE1: Cloning

which is similar to

- In- Fusion cloning (TAKARA) http://www.takara.co.kr/file/manual/pdf/pt5162-1.pdf

- Expresso cloning (LUCIGEN) https://www.lucigen.com/docs/manuals/MA108-Expresso-T7-SUMO-Cloning-Expression-System.pdf

where the linearized plasmid are direclty suppplied from the company.

Manuele Martinelli , Taq is a pretty bad polymerase because it has no proof-reading capabilities, but it is fast. A lot of thermophilic DNA polymerases exist on the market like Phusion and Pfu. It is hard to know which is better because they all say they are the best, but they all make less mistakes in amplification than Taq.

Adron Ung!

You are right! It was a typo, for High fidelity, I mean not the TAQ but different polimerases.

Up to 10Kb in my expertise

KApa Hifi (Roche)

Clone Amp (Takara)

are very good!

Sorry again for the mistake!

Manuele

Hmm, I haven't tried those polymerases. Good eh?

I am trying to do something like this right now, to clone in a retargeted SGRNA into a plasmid. But my PCR is only producing heavy junk that doesn't leave the gel well and does not migrate. The protocol and issues are described here:

https://www.researchgate.net/post/opinions_on_using_PCR_to_re-target_plasmid_sgrna_with_a_universal_sgrna_scaffold

protocol:

"2. Order two 60-mer oligonucleotides, one with the sgRNA guide coding sequence (Step 1with the reverse complement of the coding sequence. 3. Dilute each of the two 60-mers to 100 μM in water. 4. Assemble the reaction in a polymerase chain reaction (PCR) tube: Phusion HF buffer (5×) 5 μL dNTP (10 mM total; 2.5 mM each) 0.5 μL Coding sgRNA guide sequence (60-mer; 100 μM) 0.1 μL Reverse complement of sgRNA (60-mer; 100 μM) 0.1 μL pCAS plasmid 40 ng Phusion DNA Polymerase 1 μL H2O to 25 μL

Perform thermocycling with the following profile, (over 300 minute pcr!)

1 cycle 98 ̊C 1 min 30 cycles 98 ̊C 30 sec 58 ̊C 1 min 72 ̊C 10 min 1 cycle 72 ̊C 10 min 4 ̊C Hold 6. Add 1 μL of DpnI, 3 μL of the provided 10× digestion buffer, and 1 μL of water to the reaction and incubate for 6 h at 37 ̊C (or overnight). 7. Transform the DpnI-treated reaction into E. coli competent cells."

I have tried fewer cycles (21), extension time of 5:30, and high GC buffer, dmso, etc.

I also tried lower template concentrations after the DpnI reaction at 50% and 10%, but I got very low pcr yields from those.

Has anyone tried this method? Any tips/pointers?

If it would work, it means not having to obtain a unique sgrna vector for every CRISPR experiment.

CRISPR–Cas9 Genome Engineering in Saccharomyces

cerevisiae Cells

Owen W. Ryan,1 Snigdha Poddar,1 and Jamie H.D. Cate1,2,3,4

1

2Energy Biosciences Institute, University of California, Berkeley, California 94720; Department of Molecular and

3Cell Biology, University of California, Berkeley, California 94720; Department of Chemistry, University of

California, Berkeley, California 94720

This protocol describes a method for CRISPR–Cas9-mediated genome editing that results in scarless

and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results

from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a

uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule

that is used to repair the chromosomal DNA damage by homology-directed repair. For target specif-

icity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA

sequence within the sgRNA cassette. This CRISPR–Cas9 protocol includes methods for (1) cloning the

unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides,

and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically

facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial

polyploid isolates. Therefore, this CRISPR–Cas9-based DNA integration protocol is achievable by

virtually any yeast genetics and molecular biology laboratory.

I got the above working by diluting the pcr template from 40ng to 10 ng

Dear Adron Ung

in case you are interested to test those polimerase attached you can see a file with the results of a plasmid amplification by PCR.

ciao Manuele

Taq is the "Budweiser" of polymerases. I only use it for 'dirty' PCR with manganese to introduce random mutations.

Amy Johnson , I have always wanted to use taq to repeatedly amplify green fluorescence protein so I could make it randomly red or orange, and so that I could feel like screwing up a gene was an accomplishment.

And now let us raise a pipette of taq polymerase, stand and give forth our Solemn Praise, for RIP Kary Mullis, inventor of PCR, acid damaged wannabe rock star, Nobel Laureate and fan of striptease. 1944-2019

Adron Ung I could really use a red minisog, but it would take a hell of a lot of mutagenesis to make it though months of work at least - i need my gfp like sequences under 150 bp like minisog

Adron Ung

https://www.uniprot.org/uniprot/P42212

Mutagenesisi65S → G in EYFP; leads to yellow fluorescence

lots of small changes and even SNPs in the GFP lead to color phenotype changes

someone went and did this for the large GFP gene, but not minisog? would be much easier on such a short sequence, perhaps

Amy Johnson , you could totally do it. I don't know when it would work but I have a good feeling about this. These scientists used error prone PCR to make a better red fluorescent protein. They also randomly obtained a green fluorescent protein from a red fluorescent protein template.

They used the Diversify® PCR Random Mutagenesis Kit to make it happen. Clearly you need taq to make the mistakes. Varying an unequal concentration of dNTPs does help with Taq making mistakes, but if you overdo it you can get a nonfunctional protein.

Taq has a habit of adding 3' terminal A's to both strands of the genes it amplified making it a good candidate for TA-cloning for which there are kits that you can buy. Once you get Taq to amplify your gene, visualize it on an agarose gel. Get it to screw up a little bit. TA clone your constructs into a linearized TA cloning vector.

Transform that construct into protein expressing cells, and plate the cells on the appropriate antibiotic + IPTG LB plate encoded on the TA-cloning vector. If any of those colonies look red, take that plasmid and grow it up in a cell line that you can use to harvest the plasmid. I would use like 100 mL of LB to harvest the plasmid because my sequencing facility gives me terrible data if I obtain a low concentration of plasmid DNA.

Oh, if minosog requires a certain substrate, that substrate should be on the plates, as well. The primers don't make the mutation, Taq does. Where it does doesn't matter so long as you get a red colony.

Terskikh, Alexey V., et al. "Analysis of DsRed mutants Space around the fluorophore accelerates fluorescence development." Journal of Biological Chemistry 277.10 (2002): 7633-7636.