12 replies to this topic

[Curtis]

Hi,

I need to delete about 33 bp of a gene, corresponding to 10 aa on its protein product. The gene is already cloned to pFLAG. what is the ordinary and common method? to do that?

Thanks

[pDNA]

doing "invers" PCR, design primers that amplify your plasmid without the 33 bp. You will end up with a linear PCR fragment (that has to purified from your template plasmid containing the 33 bp by DpnI digestion and/or gel purification) ...this linear fragment can than be ligated and transformed.

Regards,
p

Curtis, on 06 October 2010 - 09:06 AM, said:

Hi,

I need to delete about 33 bp of a gene, corresponding to 10 aa on its protein product. The gene is already cloned to pFLAG. what is the ordinary and common method? to do that?

Thanks

[Curtis]

thanks but how to design those primers?

[bob1]

20ish bp on each side of the sequence to be deleted is the specific primer part.  You need to add restriction sites to the primers so that they can be re-ligated after amplification.

[pDNA]

i would not add RE sites because that can mess up your reading frame ...you can phosphorylate your PCR product and ligate the blunt ends.

Regards,
p

bob1, on 06 October 2010 - 03:47 PM, said:

20ish bp on each side of the sequence to be deleted is the specific primer part.  You need to add restriction sites to the primers so that they can be re-ligated after amplification.

[bob1]

Adding a kozak sequence before the ATG but after the RE site, should fix that problem.

[Curtis]

guys, can you please tell me why DpnI digests the template plasmid only, and not the new PCR product? Is it because plasmid is always methylated at some areas? PCR product does not have methylated sites? sorry, my knowledge is still basic about this.

Do we have to use DpnI? can we continue without it? and phosphorylation is to give 3' end overhang, right? why do we have to do that at all? for better ligation?

I found another method in here that does 2 steps of PCR. It doesn't use DpnI:

http://www.methods.i...R_splicing.html

[pDNA]

you are right ...plasmid DNA isolated from most E. coli strains is indeed methylated ...PCR products are not. DpnI does only cut methylated DNA ...therefore your template is degraded ...and your background is reduced (unmodified plasmid DNA without the deletion).

You can gel purify your PCR product instead ...but most of the time both, DpnI and gel purification, reduce the background efficiently. But for sure you can go on without the DpnI-step.

If you have blunt ends you will have to phosphorylate your PCR product before ligation since the 5'phosphate groups are lacking. If you add stick ends to your primers and digest your PCR product you do not need to phosphorylate.

Regards,
p

[Curtis]

thanks so much pDNA, if you could just answer one more question I hope God sends you to heaven directly  

The T4 ligase that I have is from Fermentas company and on the datasheet it doesn't say anything about phosphrylation with kinases or any other type of phosphorylation. The protocol reads as:

Self-circularization of Linear DNA

       1. Prepare the following reaction mixture:
          Linear DNA 10-50 ng
          10X T4 DNA Ligase buffer 5 µl
          T4 DNA Ligase 5 u
          Water, nuclease-free to 50 µl
          Total volume 50 µl
       2. Mix thoroughly, spin briefly and incubate:
          – sticky-ends for 10 min at 20°C,
          – blunt-ends for 1 hour at 22°C.

lets say after PCR with pfu we end up with a blunt-end product. Why do we have to phosohrylate the ends or phosphorylate the primers at all if T4 ligase can easily self-ligate it?

[Curtis]

However if you check the link that I gave in my previous post their method is very quick also. They do 2 PCR steps. the second step self-ligates the PCR ends from first step. It sound very easier than ligation and phosphorylation!

Edited by Curtis, 12 October 2010 - 07:46 AM.

[rkay447]

Curtis, on 12 October 2010 - 07:46 AM, said:

However if you check the link that I gave in my previous post their method is very quick also. They do 2 PCR steps. the second step self-ligates the PCR ends from first step. It sound very easier than ligation and phosphorylation!

Curtis~
The protocol you posted is not a ligating step but rather you are amplifying each side of your gene upstream and downstream of the desired deleted area and then using these two pieces as template in the second pcr to create the full length gene with the deletion.  The two pieces do not ligate per se but they do have small overlapping regions so the polymerase can use both to create the final product.  At the end of this step you are going to have a pcr product that you need to ligate into a vector as normal.  Normally, when you digest a piece of DNA with restriction digests, it leaves a phosphate group on the end.  You must have a phosphate on at least one end (insert or vector) in order to get ligation.  Usually people use the phosphate on the insert (that is present after restriction digest) and that way you can dephosphorylate the vector to prevent recircularization.  No phosphate, no ligation.  But, it you order oligos and don't plan on digesting, you must add the phosphate with a kinase.

[pDNA]

Hi Curtis,

   its god speaking, directly from heaven ...no, i'm just joking

The method that provided in your link is also known as stich PCR. The protocol sounds very easy but in reality it is not that straight forward as it looks like. I think re-circularization of a PCR product is faster and easier since you only need one pair of primers ...but thats my personal opinion ...other people may think different and might have different experience.

The thing about ligation and phosphorylation:
If you ligate for example a PCR product in a vector that was opend by a blunt-end cuter you do not need to phosphorylate since the vector will have the phosphate groups that are necessary to create the phosphodiesther bond. If you are just re-ligating a PCR product it is essential to phosphorylate, otherwise you will end up with NO colonies except the background-colonies created the template plasmid. Do phosphorylate even if it is not stated in the manual of Fermentas ...believe me and you will live happily ...in prosperity  

Good luck!

Best regards,
p

    

Curtis, on 12 October 2010 - 07:46 AM, said:

However if you check the link that I gave in my previous post their method is very quick also. They do 2 PCR steps. the second step self-ligates the PCR ends from first step. It sound very easier than ligation and phosphorylation!

[Curtis]

thanks so much guys, you got your tickets to heaven