Restriction Digestion!! (View forum version)



minnu

Posted 23 December 2010 - 07:18 PM

Hi all!
Iam trying to insert 2 genes to a bicistronic vector.Now I need to digest my vector with restriction enzymes, Is it possible to digest it with 4 enzymes one time
with buffer(don't know which buffer to use!)my vector is pbicmv1 and enzymes I need to use are Bgl2,EcoR1,Hind3,EcoR5.Any suggestions please,,,

perneseblue

Posted 24 December 2010 - 08:55 AM

Yes, can you use 4 enzymes in the same digest, provided

1 - all enzymes can work in the same bufer
2- the total volume of the restriction enzymes combined must be 5% or less of the volume of the restriction digest.
So for a 100ul digest, you can only use a total of 5ul of enzymes.

All RE come in a glycerol storage buffer. While this glycerol preserves the RE, the glycerol also inhibit the enzymes activity.

For alot of useful information about restriction enzymes go to NEB technical guide, on NEB's website.
Buffer finder

From the NEB website Buffer 2 +BSA would be suitable for this quadruple digest. You should also consider digesting for a longer than normal time.

However I am wondering about your ligation strategy. Why do you need to cut the vector with 4 RE?

minnu

Posted 24 December 2010 - 07:46 PM

Yes, can you use 4 enzymes in the same digest, provided

1 - all enzymes can work in the same bufer
2- the total volume of the restriction enzymes combined must be 5% or less of the volume of the restriction digest.
So for a 100ul digest, you can only use a total of 5ul of enzymes.

All RE come in a glycerol storage buffer. While this glycerol preserves the RE, the glycerol also inhibit the enzymes activity.

For alot of useful information about restriction enzymes go to NEB technical guide, on NEB's website.
Buffer finder

From the NEB website Buffer 2 +BSA would be suitable for this quadruple digest. You should also consider digesting for a longer than normal time.

However I am wondering about your ligation strategy. Why do you need to cut the vector with 4 RE?


minnu

Posted 24 December 2010 - 08:02 PM

Thank u for the reply, well ,the problem is simple,since Iam new to this field,little bit confused.
by-mistake i have added Bgl2 to the reverse primer(geneB)which will cut my second gene(geneA),since I am using a Bicistronic vector(pbicmv1).Rather than ordering a new primer I thought of 1-digesting the vector first with the enzymes,then
2-do the same with 2 genes(both are short fragments 600bp max)
3-ligate together.
what do you think, will it work? or I need to change my primer.
Thank you !:)

perneseblue

Posted 25 December 2010 - 10:12 AM

I need more information to determine if this ligation strategy will work

So I am going to make a few assumption.
I am assuming that you are going to simultaneously insert both gene 1 and gene2 into the vector.

The restriction sites on the Gene fragments are as below.

HindIII-Gene1-EcoRV
EcoRI-Gene2-BglII

I am assuming that you are going to PCR amplify both Gene1 and Gene2. You are then going to clean up those PCR fragment. After that, you are going to digest the Gene fragments using the RE
For Gene 1 it will be HindIII and EcoRV
For Gene2 it will be EcoRI and Bgl2

You are then going to gel purify each Gene fragment.

Next you are going to digest the pbicmv1 vector with EcoRV, EcoRI, BglII and HindIII.
You are then going to gel purify the vector. You will then isolate two fragments from that gel. The backbone of pbicmv1 and the CMV-enh-CMV (~600bp) fragment

After that you are going to perform a 4 way ligation.

If my many assumptions are correct, then yes, this ligation strategy should work.

However, primers are cheap. It would be simpler (and safer) to order a new primer with the correct restriction site. A restriction site site that does not cut Gene1.

minnu

Posted 26 December 2010 - 12:46 AM

Thank you for the reply.
yes,My ligation stratey is exactly like what you have said.
Any way I will try once this way and check whether it is working or not,otherwise will change the primer.Thank you for the help. i will defnitely let you know the result.