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dephosphorylated vector and PCR insert - (Nov/27/2006 )

I am reading and finding that I can't use dephosphorylated vector with the PCR constructed insert, because it lacks 5' phosphate group. dry.gif did I misunderstand something? If this is the case, how do I prevent self-ligation with my single digestion (EcoRI) reaction? thanx a lot!

-Kathy-

That's because PCR product is dephosphorylated after PCR. So, if vector has no 5'-PO4 and insert neither, ligation will never ocurr. Remember it needs both 3'-OH and 5'-PO4.

I know two ways to get it:

1. Use an insert equimolar concentration higher than vector's, to asses insert get into your phosphorylated plasmid, i.e. 4:1, 3:1 of insert:vector. Maybe plasmid could religate, but negative control in transformation will tell you this (phosphorylated plasmid). With this ratios of insert:vector it's more likely ligation ocurr between plasmid and insert, so you'll obtain several colonies ready to screen.

2. You can use dephosphorylated plasmid, but you need phosphorylate the insert/PCR product. To this, you must phosphorylate your primers, for example with T4 polynucleotide kinase, before PCR. Then, primers finish with the 5'-PO4 essential for ligation and vector will not religate.

I hope it helps.

Regards! smile.gif

-aleruiz-

I hope you don't mind, but i am just a bit unclear of the ligation strategy....

You have a PCR insert...
and a EcoRI cleaved vector.

I am mistaken here? How are the two going to get ligated to each other? The PCR insert is blunt end while the vector has a 5' overhang.

-perneseblue-

hi
the assumption you are doing is quite false as you're doing an EcoRI digestion of your PCR product, which produce the 5'P terminus you need in your reaction of ligation.
So you can CIP your vector and do the ligation as well. That's a standard strategy done in many labs.

Tip : after CIP, don't inactivate by adding EDTA, which reduce drastically the efficiency of ligation.

-fred_33-

i am really stupid in molecular biology ph34r.gif

Fred, thank you, i think i got it, if im doing blunt end ligation from PCR product then i need to phosphorylate my PCR product, but since i am doing EcoRI it creates overhang so i dont need to do anything it already has phosphate group on the 5' end. am I right? unsure.gif

pernesblue, my PCR insert has EcoRI sites on its both ends.

-Kathy-

QUOTE (Kathy @ Nov 29 2006, 12:39 PM)
i am really stupid in molecular biology ph34r.gif

Fred, thank you, i think i got it, if im doing blunt end ligation from PCR product then i need to phosphorylate my PCR product, but since i am doing EcoRI it creates overhang so i dont need to do anything it already has phosphate group on the 5' end. am I right? unsure.gif

pernesblue, my PCR insert has EcoRI sites on its both ends.

Yes Kathy. That is pretty much correct. However, if you were blunt cloning you usually phosphorylate your primers before your PCR because phosphorylation is more efficient on a single-stranded template. You can still phosphorylate your blunt double-stranded PCR product, you just need to denature your PCR product slightly with high temperature so that the ends can be easily phosphorylated as single strands. Digestion of the PCR product leaves a phosphate on the 5`end (no matter which restriction enzyme you use) that can be used for ligation.

-killerkoz17-

nothing to add to this clear answer from killerkoz17 ! smile.gif

-fred_33-

QUOTE
Yes Kathy. That is pretty much correct. However, if you were blunt cloning you usually phosphorylate your primers before your PCR because phosphorylation is more efficient on a single-stranded template. You can still phosphorylate your blunt double-stranded PCR product, you just need to denature your PCR product slightly with high temperature so that the ends can be easily phosphorylated as single strands. Digestion of the PCR product leaves a phosphate on the 5`end (no matter which restriction enzyme you use) that can be used for ligation.


wow! thank you! biggrin.gif

-Kathy-