Gene cloning question

[vidya790]

Hi RNAse is nothing but Ribonuclease which actually cleaves a phosphodiester bond between any two ribonucleotides and also degrades RNA into smaller components which is mostly helpful in prokaryotic processes and play a important role in many biological processes.

[kk]

I have a few besic questions due to I am simply lazy to look up old lecture books.

Before ligation of an insert into a terget vector, the target vector is treated with phosphatase, to remove WHICH phosphate group? The one at the 3'-end, 5'-end, or both?

When a restriction endonuclease cuts a double stranded DNA, which newly formed end will retain the phosphate group from the phosphodiester bond? Is it the 3' or the 5'-end?

THANKS A LOT!!!

[JackBean]

I have a few besic questions due to I am simply lazy to look up old lecture books.

You shouldn't say that

Before ligation of an insert into a terget vector, the target vector is treated with phosphatase, to remove WHICH phosphate group? The one at the 3'-end, 5'-end, or both?

There is only one, so pick, which one is it

When a restriction endonuclease cuts a double stranded DNA, which newly formed end will retain the phosphate group from the phosphodiester bond? Is it the 3' or the 5'-end?

See above

Regarding the Kozak' sequence, the note was right, you should not leave it in the midlle of transcript, otherwise transcription will statr also there (e.g. I saw it right yesterday in Invitrogen's manual;)

Regarding spacing AAs, depends on where in the structure are the ends. If they are exposed to surface, any (=zero) AAs can work, on the other hand, if they will be inside, it may cause misfolding, if there were not enough of spacing AAs

[kk]

Thanks JackBean! I am aware that there is one phosphate per nucleotide. I meant:

Restriction endonucleases: when they cut which newly formed end retains the P group, the 3' or the 5'?

Shrimp alkaline phosphatase: from which end does it have the ability to remove a P group, the 3' or the 5' or both?

And actually one more thing I don't get: at the NEB website, for BamHI it says "Heat inactivation: No". What does it mean? No way to inactivate it? No need to inactivate it - why?

[JackBean]

Shimp AP? What is that?
just 5 seconds to type it and... tradaaa
http://www.fermentas.com/catalog/modify ... phosph.htm
Description
The Shrimp Alkaline Phosphatase (SAP) catalyzes the release of 5'- and 3'-phosphate groups from DNA, RNA and nucleotides. Also this enzyme can remove phosphate groups from proteins.

(usually the phosphatases don't care much from where they are removing the phosphate, just do it

Indicates whether or not the enzyme can be heat inactivated. Enzymes are first tested by incubation at 65°C for 20 minutes; any enzyme not inactivated at 65°C is then tested byincubation at 80°C for 20 minutes.

[MrMistery]

this shrimp alkaline phosphatase is just a type of CIP. They now have a million of them, but they do all the same thing. Personally I rarely need to use it. My problem is usually lack of colonies not high background.

@kk
obviously if you heat a protein enough it will get inactivated due to misfolding, but if you boil DNA it will also cause nicks. Generally you shouldn't put the DNA at above 85 degrees celsius. NEB tests their enzymes at 65 and 80, like JackBean said http://www.neb.com/nebecomm/tech_refere ... vation.asp

[kk]

My current problem:

I want to ligate synthetic oligo to a plasmid (I ordered 2 single stranded DNA oligos, which are reverse complementary of each other except their ends, I hybridized the two oligos, now I have an insert with both ends sticky).

Should I use alkaline phosphatase (CIP, SAP) to remove 5'-phosphate from the digested plasmid vector (to prevent self ligation)? In other words: does the synthetic oligo contain 5'-phosphate (which will then be needed for ligation)?