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Big insert to put back into the vector


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#1 ups

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Posted 17 February 2009 - 05:04 AM

Dear all,

Please help me!!!


I have a vector in which there is a gene of interest and a tag. I want to remove the gene and leave the empty vector but with the tag in, so I can clone my own gene of interest in it. There is no restriction enzyme site at the end of my vector, but there is one within the tag-gives blunt ends. So I would cut 33 bp from the tag.

Can I put them back using the reverse primer and get a normal PCR product?

What should I do?

Thank you in advance,

Ana

Edited by ups, 17 February 2009 - 05:05 AM.


#2 T C

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Posted 17 February 2009 - 05:47 AM

Hey

How big is the tag?

You can always engineer another site in front of the tag by PCR and either do a PCR in two steps (one for tag and one for gene) or do an overlap PCR. There are easier ways of doing it but the advantage here would be that in future you just need to clone all yr genes with this unique site at the end and you have tag ready. I would put something like HindIII or Eco RI which can also be used for other vectors.

Best
TC

#3 perneseblue

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Posted 17 February 2009 - 07:10 AM

alternatively..

how big is your tag?

You can use a large primer to add the tag directly to your new gene when you PCR amplify it. Just create a the tag sequence to the reverse 3' primer. (Don't forget the stop sequence)

As for the vector, you can then cut behind the tag sequence.
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#4 ups

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Posted 17 February 2009 - 08:42 AM

Dear all the vector is 12532bp, the gene that I want to take out is from 1 to 2999. at pos 1 is Eco R1, at 2999 there is nothing until 3011-but I cannot use as it is BamH1 and I have BamhI site in my interest gene that I want to put in, and at 3032 there is SmaI which I would like to use but this means that I have to put back 33bp from the vector (which is the seguence for a clivage site and a bit of the tag itself).

So I can just add the seguence that I cut to my reverse and it should work?

The tag is big, around 290bp, and it is from 3017 to 3295.

Thank you,

Ana

PS: By the stop codon you mean the one in the gene of interest right?

Edited by ups, 17 February 2009 - 08:47 AM.


#5 perneseblue

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Posted 17 February 2009 - 08:57 AM

PS: By the stop codon you mean the one in the gene of interest right?

Yes. I mean the stop codon at the end of the tag. Which no longer is applicable in this case as the tag is large.

So I can just add the seguence that I cut to my reverse and it should work?


Yup, it should be okay. The primer as long as 100bp can be use for PCR.

Hmm... there are restriction enzymes that produce compatible overhangs. BamHI, BglII and BclI (dam sensitive) all produce compatible overhangs. So you can ligate BamHI cut DNA with BglII cut DNA. The restriction site will be destroyed so it is a one way journey.
May your PCR products be long, your protocols short and your boss on holiday

#6 ups

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Posted 17 February 2009 - 09:11 AM

PS: By the stop codon you mean the one in the gene of interest right?

Yes. I mean the stop codon at the end of the tag. Which no longer is applicable in this case as the tag is large.

So I can just add the seguence that I cut to my reverse and it should work?


Yup, it should be okay. The primer as long as 100bp can be use for PCR.

Hmm... there are restriction enzymes that produce compatible overhangs. BamHI, BglII and BclI (dam sensitive) all produce compatible overhangs. So you can ligate BamHI cut DNA with BglII cut DNA. The restriction site will be destroyed so it is a one way journey.



Sorry for my persistence, but just to have something clear:

I can introduce a big sequence (33bp) in my primer, although there is no seguence that matches it in my template , my templete is a pcDNA which I use to PCR my gene of interest and than put it in the clean vector.

Is that correct? So at my reverse primer I will something like: 3' 15-18bp gene on interest-33bp which includ missing seguence-restriction enzyme site (half for Sma1)-and maybe 1-2 nucleotides 5'?

Thank you

#7 perneseblue

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Posted 17 February 2009 - 09:23 AM

yes.

3' 18-25bp gene on interest-33bp (which include missing sequence)-restriction enzyme site-6 guard nucleotides 5'
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#8 ups

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Posted 17 February 2009 - 11:15 AM

Sorry another Q: when I calculate the TM I do not consider the insert that does not have correspondance within my seguence?


Thank you

#9 perneseblue

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Posted 17 February 2009 - 11:54 AM

Sorry another Q: when I calculate the TM I do not consider the insert that does not have correspondance within my seguence?


Yes, quiet right. Only the DNA sequence that actually binds to the template is used for the tm calculations. If primer is 100bp long and 23bp anneals to the template. The tm calculated on the 23bp.

Also you might want to use the enzyme XmaI rather than SmaI for the digest. Both restriction enzymes cut the same DNA sequence, but XmaI has two advantages over SmaI. XmaI 5' overhang while SmaI is blunt end. Thus the ligation is easier. Next XmaI cuts at 37 C while SmaI at 25 C. Thus you can use XmaI and EcoRI in a double digest in NEB buffer 4.
May your PCR products be long, your protocols short and your boss on holiday

#10 ups

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Posted 17 February 2009 - 12:10 PM

Sorry another Q: when I calculate the TM I do not consider the insert that does not have correspondance within my seguence?


Yes, quiet right. Only the DNA sequence that actually binds to the template is used for the tm calculations. If primer is 100bp long and 23bp anneals to the template. The tm calculated on the 23bp.

Also you might want to use the enzyme XmaI rather than SmaI for the digest. Both restriction enzymes cut the same DNA sequence, but XmaI has two advantages over SmaI. XmaI 5' overhang while SmaI is blunt end. Thus the ligation is easier. Next XmaI cuts at 37 C while SmaI at 25 C. Thus you can use XmaI and BamHI in a double digest in NEB buffer 4.



:angry: Thank your for answer

Things aren't simple though:

I have multiple isoforms(different splice variants)
2 have no site for SmaI within the site but have for BamH1
6 have sites for Sma1 and BamH1

So for the first two I was thinking of using EcoR1 at 5' end and SmaI ( or now XmaI), and I shell put overhanging nucleotides (A,T) 1-2 on both sites as I can cut and not clive my gene of interest, or shoul I put more?

But for the other 6 I guess I must use Sma1 at 3' end and add no overhanging nucleotide(?) so I wouldn't need to clean it with Sma1 at that end, otherwise cuts my gene-so I would prefer blunt end.


What do you think?

PS: so when calculatingTM, I even do not consider the restriction site that I insert in my primer, corret?

Thank you for your help:)

#11 ups

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Posted 17 February 2009 - 12:16 PM

I forgot to ask smthing:)

If I have about 33-34 bp which do not match anything, how many should I add from my seguence so it would work fine ? I had about 15bp but the Tm is very low and does not match the forward (20compared to 58), how long can the primer be, as you can image with only 15bp matching, if I put the whole Sma1 seguence it is 51bp:), and a couple of overganging...


Thank you,


Ana :angry:

Edited by ups, 17 February 2009 - 12:21 PM.


#12 perneseblue

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Posted 17 February 2009 - 12:34 PM

XmaI needs at least 3bp flanking each end of the restriction site before the enzyme will cleave the restriction site efficiently. I would use at least 5bp to be on the safe side.

the primer can be as long as 100bp, the maximum that most companies will sell you. The tm of both primers must be as close as possible. As you have noted 15bp is too short. Make it longer, increase the length of the sequence until the tm of both forward and reverse primers are the same. I would aim for a tm of 58 Celsius.

Could you rephrase "If I have about 33-34 bp which do not match anything, how many should I add from my seguence so it would work fine ??". I am not certain what you mean. (Below is what I think you mean)

I assume that this 33-34bp block must be included and is inert as far as PCR goes. AKA this block doesn't bind to your template. The length of the sequence that does bind to the gene template is dependent on the tm rather than the length. I would aim for tm of 58 Celsius for all my primers. Thus a the section of the primer that bind to an AT rich template would be longer than a primer that bind to a GC rich template.
May your PCR products be long, your protocols short and your boss on holiday

#13 ups

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Posted 17 February 2009 - 02:51 PM

Yes,

That was what I meant, 30bp which I must put in but do not match my template.

But what about blunt ends? can I used them without adding any overhanging pb? so I wouldn't need a clean up afterwards


Thank you in advance :angry:

#14 perneseblue

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Posted 17 February 2009 - 03:25 PM

Yes,

That was what I meant, 30bp which I must put in but do not match my template.

But what about blunt ends? can I used them without adding any overhanging pb? so I wouldn't need a clean up afterwards


Yes. If you used blunt end cloning. You don't need to add any overhanging bp. However blunt end cloning is more difficult that sticky-end cloning. So whenever possible I would try to avoid blunt end cloning.

Your PCR amplified gene already has a EcoRI site on the 5' end. So you have to clean up the product regardless. Thus one might as well have a sticky end XmaI on the 3' end
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#15 ups

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Posted 18 February 2009 - 05:40 AM

Yes,

That was what I meant, 30bp which I must put in but do not match my template.

But what about blunt ends? can I used them without adding any overhanging pb? so I wouldn't need a clean up afterwards


Yes. If you used blunt end cloning. You don't need to add any overhanging bp. However blunt end cloning is more difficult that sticky-end cloning. So whenever possible I would try to avoid blunt end cloning.

Your PCR amplified gene already has a BamHI site on the 5' end. So you have to clean up the product regardless. Thus one might as well have a sticky end XmaI on the 3' end



Sorry for all these quenstions, I have another Q: would it be more helpful if I would add after the 33bp non matching seguence, some bp that would match my template just after the gene of interest stops? :)

Sorry for the questions:(




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