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Gene cloning question

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Re: Gene cloning question

Postby vidya790 » Wed Sep 16, 2009 9:22 am

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.
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Postby kk » Wed Oct 07, 2009 3:08 pm

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!!!
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Re:

Postby JackBean » Fri Oct 09, 2009 7:23 am

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

You shouldn't say that :-D

kk wrote: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 ;)

kk wrote: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 ;)
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Postby kk » Fri Oct 16, 2009 10:03 am

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?
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Postby JackBean » Fri Oct 16, 2009 12:13 pm

Shimp AP? What is that? :-D
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.
http://www.biolib.cz/en/main/

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Postby MrMistery » Sat Oct 17, 2009 1:43 am

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
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Postby kk » Thu Sep 08, 2011 8:39 am

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)?
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Re: Gene cloning question

Postby mohajeri » Thu Feb 06, 2014 10:01 pm

I want to clone a human gene in pET-21a, and transfer to E.coli. I clone the gene in Nde 1 site that it’s sequence is CA/TATG, My question, is The ATG of Nde1 act as a start codon in result I must delete The ATG of my sequence or I can use the ATG of vector in Nde1 site as a start codon? (NOTE: My gene is eukaryotic)
Is there anyone make a sugestion?
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Postby JackBean » Fri Feb 07, 2014 9:38 am

You replace one ATG with another, don't you?

However if I were you, I'd be rather concerned about the start codon of the N-terminal fusion.
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