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single restriction enzyme digestion? - is it possible to insert PCR product? (Jun/07/2006 )

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i have another question...i want also to insert my PCR product before the tag where there is no MCS there is only one HINDIII site....so i want to insert PCR product there.....i lose directionality right? i cant make PCR product with HindIII sites at both ends... unsure.gif ...maybe i should ligate into T-vector....im searching the web about this but want to hear your opinions.....what options do I have??? thanx a lot.

-Kathy-

1. yes you'll lose directionality - why you can't make pcr product with HindIII at both ends?
2. you could also try to make a second restriction site by site directed mutagenesis
3. check that you don't disturb any important features of the vector, if you use some other point of insertion for the dna than the MCS

-Kersten-

QUOTE (Kersten @ Jun 7 2006, 11:15 PM)
1. yes you'll lose directionality - why you can't make pcr product with HindIII at both ends?
2. you could also try to make a second restriction site by site directed mutagenesis
3. check that you don't disturb any important features of the vector, if you use some other point of insertion for the dna than the MCS


thanx a lot for the reply, i mean i can make HindIII at both ends but i still lose directionality no?... unsure.gif no problem at making two resitriction sites on PCR but the problem is how i make sure it is inserted in the right direction. i hope im not disturbing the vector by that but its really important to me if i can insert cDNA before the tag.....

-Kathy-

Okay, got it... there is no way I know how to prevent losing the directionality if you use only on restriction enzyme. what about my suggestion to introduce a second one by site directed mutagenesis? are there any features like antibiotic resistance that you interfere with or something like that? are there no other vectors for your task maybe?

-Kersten-

well assuming the direction of insertion is 50/50, you mayhave to screen more clones.
I've done that for my purposes.
Can you check the direction by restriction analysis?
If not, you may consider an isocshizomer of hindIII. The ligation HIND/isoshizomer will kill the site and so you may check where is the HINDIIIfull site

-fred_33-

I dont think it is problem losing directionality during insertion.
you might check direction by PCR using one primer of you PCR products and one on vector.
just like fred said, 50% of insertion is your favour result...

-rshi-

thanx a lot everyone for your valuable suggestions!

Kresten, you mean site directed mutagenesis of the vector....i think it may be possible but time consuming no? i never did site directed mutagenesis.....so i might leave it as last option if others dont work....thanx for the idea!

Fred, ill think about restriction enzyme check....you are right i just can get 50%.....

QUOTE
you might check direction by PCR using one primer of you PCR products and one on vector.


you mean PCR check of the colonies right?

-Kathy-

sorry just making sure....i should put HindIII site on both forward and reverse primers right?

-Kathy-

Yes, that`s correct. The PCR product can then be inserted into your HindIII site in the vector.

smile.gif

-chalet2-

QUOTE (Kathy @ Jun 8 2006, 07:45 PM)
sorry just making sure....i should put HindIII site on both forward and reverse primers right?

you even can blunt digested vector and PCR product for ligation, sure the ligation efficiency is lower and you should screen more but it still work.

-rshi-

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