5 replies to this topic

[3T3]

I am trying to digest a vector with SmaI and EcoRI for cloning......the cutting sites are very close and share one G base.....NEB suggest sequential digestion for this two enzymes.....but I can't figure out how to do it as both enzymes use different salt in their buffer....Please help me if anyone have any experience in this regard....

Regards,
3T3

Edited by 3T3, 23 June 2011 - 11:22 AM.

[Bomber]

digest the plasmid first with either enzyme, purify it (e.g. pcr purification kit or something) and then digest with second enzyme using the appropriate buffer.
i wonder if you can successfully digest though since apparently the recognition site is shared

[leelee]

If I'm reading this right, and when you say that they share a G base that you mean the recognition sites overlap by a G, then what you are trying to do is impossible.

SmaI will not cut DNA without at least 2, preferably 3, bases either side of the recognition site.
EcoRI needs at least 1.

Edited to add- see NEB site here:
http://www.neb.com/n...nucleotides.asp

Edited by leelee, 23 June 2011 - 07:50 PM.

[donny]

With reference to leelee's comment, the link given is for short oligos. For a vector, this link is more apt because linearised plasmids would have one longer flanking end that may be sufficient for digestion. However, since SmaI is not listed in the link, it may "require 6 base pairs on either side of their recognition site to cleave efficiently" as stated in the link. Therefore the long flanking end may or may not be enough for cleavage of the SmaI site.

When NEB suggests sequential digestion, it is usually to minimise star activity. In this case, it may be due to the difference in digestion temperature as well (25C for SmaI and 37C for EcoRI). Since they both have 100% activity in NEBuffer 4, I suggest you try double digestion in the buffer first (first at 25C then 37C) and see if it works [I've worked against the sequential digestion advice with no problem ]. But since EcoRI seems to have less problem cleaving a site with short flanking ends, it may be better to digest with SmaI at 25C, then add EcoRI and digest at 37C. This way, SmaI would be digesting the plasmid with two long ends flanking the restriction site and EcoRI will be left to do the "tough work" of digesting the linearised vector with the EcoRI site flanked by 3bp. Make sure the glycerol concentration is kept below 5%.

[leelee]

My bad, posted the wrong link.


Let us know if it works, 3T3. I'd be interested to see  

[3T3]

Hello Everyone....Thanks a lot for ur comments....I am digesting first with SmaI and EcoRI.....so SmaI will have sufficient bases after its cutting site and EcoRI is more flexible....The sequence is like CCCGGGAATTC-----I have digested the vector with these two enzymes and I think it has been digested but I don't get clones now....I see colonies in plate and no colonies in negative control.....I inoculated the colonies in LB+Amp for 6-7 hours....spin down to get pellet and resuspended the pellet in 1 ul and did PCR with that....but the problem is that i don't see bands...I only see some very intense bands in the primer dimer area....(its not the primer dimer....coz I also had a sample without template, so that I can see the primer dimer in gel and it shows a faint primer dimer). Positive control of PCR had band in right position and I did ligation overnight.....Can anyone suggest what is going on...

Regards,
3T3