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Changing the vector

cloning

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55 replies to this topic

#46 Bio-Lad

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Posted 01 October 2014 - 03:20 AM

Again though, if you are already getting a visible band using your other conditions, simply doing a TOPO cloning may be the way to go.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#47 RNA woman

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Posted 01 October 2014 - 11:08 PM

For the Topo cloning. It is written as note to not use 5 phosphates primers for PCR that PCR product synthesised will not ligate. I don't understand why? I will remove the primers with the column and the PCR product it self will not have 5' phosphates. 



#48 phage434

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Posted 02 October 2014 - 03:22 AM

Your PCR product will have 5' phosphates if the primers have those phosphates. I was unaware of that issue with TA cloning, since I never use it. You can remove the 5' phosphates with shrimp alkaline phosphatase or antarctic phosphatase. Likely you can directly add 1-2 ul of SAP to your PCR reaction following PCR, without purification. AAP will require adding its buffer, which contains zinc.



#49 Bio-Lad

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Posted 02 October 2014 - 03:32 AM

I never knew that either. I was just looking it up. You learn something new every day!

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#50 RNA woman

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Posted 07 October 2014 - 04:40 PM

Bio-Lab,

I found something here. The primers that you suggested and I used to amplify the gene. have EcorV and NdeI not NheI restriction site.

I found also EGFR has two NdeI sites in the meddle of its sequence. Could you confirm please?


Edited by RNA woman, 07 October 2014 - 05:08 PM.


#51 Bio-Lad

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Posted 07 October 2014 - 04:59 PM

Crap, yes it appears I put the wrong one in there. Apologies. I'm afraid I'm not perfect...yet.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#52 Bio-Lad

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Posted 07 October 2014 - 05:04 PM

You may be able to get around this by doing a blunt TOPO cloning and using one of the sites in that vector's MCS with EcoRV. Otherwise you'd need to reorder the reverse primer. Again, my apologies.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#53 RNA woman

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Posted 07 October 2014 - 05:05 PM

No worries! I am learning good things everyday:) May be you can teach me how to ligate blunt PCR product to blunt vector and keep the directionality of the gene ( in correct ordination)?

I am thinking of two ways to do this:

1- to use EcoV for the left side that will give blunt end. and find other blunt side at the right side and do blunt ligation then the directionality problem appears.

2- after ligation reaction that where I am now. gel purify what I think correct size then use it at the transformation step.

What do you think?


Edited by RNA woman, 07 October 2014 - 05:06 PM.


#54 Bio-Lad

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Posted 07 October 2014 - 05:11 PM

You could do that but you'd want to dephosphorylate the vector after digesting it or you'll get a ton of colonies from the vector ligating to itself. After getting colonies, you'd want to do a digest of several clones that will tell you in which direction the onset went. It should be about 50% for each direction.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#55 RNA woman

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Posted 07 October 2014 - 05:13 PM

what about option 2? gel purify the correct size before transformation?



#56 Bio-Lad

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Posted 07 October 2014 - 05:19 PM

Well it's not a bad idea to gel purify the correct band from your PCR, especially if you see several bands.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/






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