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need help in my Fse1 restriction ezyme - (Nov/10/2009 )

hai..

could anyone help me in resolving this problem?
i had amplified Kan resistant gene with Fse1 restriction site(ggccggcc) at both end, I ligated in pGEMT vector and send for sequencing. Results from sequenceing showed that the both restiction site forward and reverse present. Thus i proceed with restriction enzyme Fse1 to cleave the Kan resistant gene in order to ligate it into my gene of interest (I use single digestion using Fse1 since that was the only option i had to carry out the cloning procedure). However after i treat the vector with restiction ezyme for 1hour and 3hours in 37C and run gel i could only observed a single band at 3800bases (pGEMT 3000bases+ Kan gene 800bases).

If im not mistaken the sigle band mean that the vector is in linearlized form, as compared to my contro(vector with untreated with restriction enzyme) would have 2 or more since it have supercoiled form.

Which mean that the restriction enzyme only act towards 1 site, but doesnt cleave at another site. Can anyone help me to solve this problem?y is it the ezyme doesnt cut at both site?What should i do?

protocol for RE

Vector-5ul
10Xbuffer 4-2ul
BSA-0.5ul
restiction enzyme-0.5ul
ddh2O-12ul
total-20ul

-sanuzi-

Hi there,

I just checked FseI on NEB website (don't know if you are using NEB enzymes):

dam methylation: Not sensitive
dcm methylation: Impaired by some combinations of overlapping

You need to check if this is the case. In normal e. coli you would get dam and dcm methylation

Dcm methylase–methylation at the C5 position of cytosine in the sequences CCAGG and CCTGG

So if your sequence if followed by agg or tgg, the restriction site might be methylated.

In this caase you have to change to a E. coli strain without methylation like NEBs strain 2925

dam-/dcm- Competent E. coli


Catalog # Size Concentration Price Qty
C2925H 20 x 0.05 ml/tube $216.00
C2925I 6 x 0.2 ml/tube $166.00

* Grow plasmids free of Dam and Dcm methylation
* Free of animal products
* T1 phage resistant (fhuA2)
* Transformation efficiency: 1-3 x 106 cfu/μg pUC19 DNA
* Activity of nonspecific endonuclease I (endA) eliminated for highest quality plasmid preparations
* K12 Strain

Description:
Methyltransferase deficient chemically competent E. coli cells suitable for growth of plasmids free of Dam and Dcm methylation. Note that dam- strains are not recommended as a host for primary cloning/ligation. The dam mutation can result in an increased mutation rate in the cell and a reduction in the transformation efficiency. DNA should be maintained in a dam+ strain unless there is a specific need for DNA free of Dam or Dcm methylation.

Hope this helps,

Stardust

By the way, NEBs FseI is only 2 U/l. So 0,5 l (1 U) might not be enough. How much DNA (not in l but in g) are you digesting?

-stardust-

The methylation could also be caused by overlapping dcm methylation at the front of the restriction site, so you need to check for cca or cct just before the site, as well as for tgg or agg after the site.

-phage434-

sorry, i missed that. Thank you!

-stardust-

OK- three things, which I had to learn the hard way:

1) NEB says you can store FSE1 at -20 degrees C. This is not true. It will decompose rather quickly at -20 degrees C and must be stored at -80 degrees C.
2) Do individual restriction digests (one at a time) to see which of your enzymes is cutting. The migration distance will almost always differ depending on whether it is linear or circularized.
3) FSE1 is not such an active enzyme. I'd overdigest a bit just to be safe.

Good luck!

-Nick C-