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how to design PCR primer with a tag region which use for In-frame deletion gene?

Biochemistry Molecular cloning Molecular biology gene deletion pcr

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#1 Huongnguyen.miss

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Posted 02 September 2015 - 06:12 AM

I have just started a new work - Gene deletion . I  do not understand about Tag which appear in PCR primer. 

in my work, firstly i have to design PCR primers to amplify the regions flanking the target gene. Then these flanking regions are fused together via a complementary "tag" region. This tag region should be unique, thus giving each mutant its own " barcode" for future work and identification. 

And my problem is : I dont know where tag comes frome? How i make these tag?

Any one know about this? Please help me

Thanks in advance and im looking forward your reply



#2 bob1

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Posted 02 September 2015 - 01:02 PM

You can design the tag into your primer. All you need to do is include the tag sequence in the 5' end of the primer, which will then allow amplification of the target sequence including the tag. Note that it is usually a good idea to add a few (3-6) bases 5' of the tag just to give the enzymes something to grab hold of when amplifying. That way you don't run any risk of losing the start of the tag while amplifying.



#3 Huongnguyen.miss

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Posted 02 September 2015 - 06:24 PM

that's mean i design by myself , right? but i am still confusing because i do not know  what reason or what factor would make a tag? you said add 3-6 bases in to primer so where these bases come from?

Sorry for my bad understanding :(

but i really appreciate any your help 



#4 bob1

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Posted 03 September 2015 - 12:48 AM

presumably as you are making mutants, you will know where you want the primers to be on the gene of interest. The tags - yes you will have to make them yourself, unless you have a system already in place for the sequences desired. For the tags it will be a good idea to make sure you don't have runs of any of the bases in the sequences (e.g. don't have AAAA or CCC in there). As far as I can tell from your post you are using a technique called Ligation PCR, explained by the image here: http://2012.igem.org...9c/Ligation.png

 

You don't absolutely have to add the 3-6 bases, especially if you are using the tag region to fuse the complementary ends. 



#5 Huongnguyen.miss

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Posted 03 September 2015 - 05:28 AM

yes, i am making a mutant as well as  deletion strategy.  

So i enclosed a files which i found on internet, may you see that and explain for me how they designed primers at N-terminal and C-terminal. Where should i put Restriction enzymes?

I apologize for my disturbing , i know it takes your time away but your explain would be great help for me.

Thank you so much

Attached Files


Edited by Huongnguyen.miss, 03 September 2015 - 06:21 AM.


#6 bob1

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Posted 03 September 2015 - 01:22 PM

Ok... what you need to determine is what exactly your mutants should be, i.e. which parts are the bits you want deleted?

 

Presumably these are significant deletions (20+bp) of the gene of interest. If this is the case, the 5o primer will be the first 20 or so bp of the 5' end of the gene, the 3o will be the reverse complement of the last 20 bp of the gene. The inside primers will be dependent on the location of your deletion, but using the 5o and 3i primers will amplify a fragment that stretches from 5o to 3i and the 5i will amplify in partnership with 3o producing two separate fragments in two separate reactions. These two fragments will flank (be on either side of) your target mutation.

 

In the protocol you posted, the yellow region is the bit being deleted, so as you can see they have primers that are just on the outside of the yellow region labeled 3i and 5i respectively  Their example is a little bit confusing because the OmcA gene appears to be transcribed in the opposite direction to how genes are normally depicted, which is why their 3' and 5' ends are switched.

 

I don't know how they generated the "tag" sequences, but it looks like they have just used random sequences. Note that the tags will have to be multiples of 3 bases (i.e. codons) in length. For example, they could be 18 or 21 bp, but not 20 as this would alter the reading frame once ligated together. Unless you want to later insert some sort of marker, you don't have to worry about putting restriction sites into the tags. It is not usually a good idea to insert markers into coding sequences anyway as this might well interfere with unique functions of each end of the protein.



#7 Huongnguyen.miss

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Posted 06 September 2015 - 05:02 PM

Thank you so much for your explanation. It is helpful for me to continue my work. ^^

I really appreciated all your responses 



#8 Huongnguyen.miss

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Posted 06 October 2015 - 01:04 AM

I did the cloning the fusion PCR fragment in T-easy vector. but after sequencing, it just correct 21 nucleotides ( that is primer sequence) . I dont know why it was happened? Should i do sequencing ?

Thanks so much for your response 



#9 bob1

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Posted 06 October 2015 - 11:42 PM

Which polymerase did you use for amplification in your PCR? If it wasn't Taq, did you add A overhangs for the T/A cloning to work from?



#10 Huongnguyen.miss

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Posted 28 October 2015 - 01:56 AM

i use prime star. and after PCR i add tag  to get the A overhangs. I have done 2 times for sequencing, however all colonies were totally incorrect .:( 



#11 bob1

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Posted 29 October 2015 - 02:13 PM

BLAST search some of the sequence you do have - it might be that you have an off-target PCR amplification.







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