Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
* - - - - 1 votes

Design the primers


  • Please log in to reply
10 replies to this topic

#1 Asma Safdar

Asma Safdar

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 19 August 2014 - 09:26 PM

Hi. I am new to cloning and molecular techniques. I have started Gene construction. I want to design the primers using SmaI and KpnI. Can anyone tell me the process with some example please.



#2 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,334 posts
81
Excellent

Posted 19 August 2014 - 09:52 PM

not sure what you mean...

You want primers that bind do those RE sites? to amplify a gene that is between those REsites?

Or you want primers that contain those RE sites so you can cut the amplified insert later on and digest it to ligate it into a vector with those sites?


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 Asma Safdar

Asma Safdar

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 19 August 2014 - 10:29 PM

Yeah I need to design the primers having these restriction sites so i have to cut the amplified gene and ligate into vector having these sites.



#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,784 posts
406
Excellent

Posted 20 August 2014 - 12:25 AM

Have a look here:http://www.protocol-...riction-enzyme/



#5 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,334 posts
81
Excellent

Posted 20 August 2014 - 12:44 AM

Yeah I need to design the primers having these restriction sites so i have to cut the amplified gene and ligate into vector having these sites.

 

I see.

Its pretty straightforward to be honest.

This will already help you out a lot: http://www.addgene.o...ls/PCR_cloning/

 

Have a look here:

Is it just me or is there nothing in your post?


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#6 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,784 posts
406
Excellent

Posted 20 August 2014 - 12:53 PM

Nope not just you, I pasted a link in, but for some reason it didn't show up... will fix.



#7 Asma Safdar

Asma Safdar

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 20 August 2014 - 09:13 PM

Thank you very much ffor your kind replyies it really helps me alot. 

I can design primer of gene like this.. GGGGTACCTTA20 bp of Reverse compliment of GOI-Reverse Primer 

                                                           CCCGGGATG pb of GOI-Forward Primer

 

one more question please....One of my labmate design forwrd primer for me like this, CCCGGGacaacaATG pb of GOI-Forward Primer. I can't undrstand the function of acaaca. Can anyone please tell me??

 

 



#8 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,334 posts
81
Excellent

Posted 20 August 2014 - 09:51 PM

What are the restriction sites? Are those the red ones?

If yes: you better always have a few basepairs (2-3-4-5-6) extra next to the restriction site so the RE can cut it more efficiently.

So your primers are not all ok!.

you have GG next to the restriction site for the reverse, which is good... check this website:

https://www.neb.com/...f-dna-fragments

(so you see for knpI its ok!)

 

 

For the forward primer: you also need those sites , however why he places acaaca on the right side from rather the left site, thats not clear for me.

The ACA ACA itself has a function in RNA, but I am in doubt why you need this for your gene...

But in your primer: they are not there! So you need to add them!

 

 

 

Check the website!

You need extra sites to have the RE cut more efficiently! Perhaps thats why he placed them there (the ACA ACA) , but not sure why he placed them to the right of your cut site..

 

 

 

 

Thank you very much ffor your kind replyies it really helps me alot. 

I can design primer of gene like this.. GGGGTACCTTA20 bp of Reverse compliment of GOI-Reverse Primer 

                                                           CCCGGGATG pb of GOI-Forward Primer

 

one more question please....One of my labmate design forwrd primer for me like this, CCCGGGacaacaATG pb of GOI-Forward Primer. I can't undrstand the function of acaaca. Can anyone please tell me??

 

 


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#9 Asma Safdar

Asma Safdar

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 20 August 2014 - 10:20 PM

Thankyou so much for your kind reply. Please can you tell me that which one is correct design???? RED Highlited are RE.

 

1) TCCCCCGGGGGAATG pb of GOI-Forward Primer-- SmaI

     CGGGGTACCCCGTTA20 bp of Reverse compliment of GOI-Reverse Primer--- KpnI

        OR

2)  TCCCCCGGGATG pb of GOI-Forward Primer

      CGGGGTACCTTA20 bp of Reverse compliment of GOI-Reverse Primer

 

Also what is Kozak sequence required for??? I am dealing with fungal pathogen. I need to apmlify gene first and silence that gene in that organism. Is there any difference to design primers for gene over expression or gene silencing?? May be that ACA ACA is related to this issue....


Edited by Asma Safdar, 20 August 2014 - 10:57 PM.


#10 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,334 posts
81
Excellent

Posted 21 August 2014 - 12:50 AM

ACA ACA does not seem like a KOZAk sequence to me...

 

But I am not sure why you would need a kozak sequence if you want to silence the gene? I am not sure what you are going to do actually..

You are amplifying the gene for what reason? What are you going to do with it?

 

Kozak sequence is needed for the start of the translation, the make it more efficient, but normally they are already provided in the plasmid, with the promoter in general..

 

The second set of primers seem good for me..

No need to have extra bases between the RE and the start of your gene.. (or end of your gene)

 

Thankyou so much for your kind reply. Please can you tell me that which one is correct design???? RED Highlited are RE.

 

1) TCCCCCGGGGGAATG pb of GOI-Forward Primer-- SmaI

     CGGGGTACCCCGTTA20 bp of Reverse compliment of GOI-Reverse Primer--- KpnI

        OR

2)  TCCCCCGGGATG pb of GOI-Forward Primer

      CGGGGTACCTTA20 bp of Reverse compliment of GOI-Reverse Primer

 

Also what is Kozak sequence required for??? I am dealing with fungal pathogen. I need to apmlify gene first and silence that gene in that organism. Is there any difference to design primers for gene over expression or gene silencing?? May be that ACA ACA is related to this issue....


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#11 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,784 posts
406
Excellent

Posted 21 August 2014 - 01:37 PM

Also note that SmaI is a blunt cutter - this makes your ligations quite inefficient and relatively difficult to perform, so if you can, avoid Sma1 and find another site that produces sticky ends.






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.