Login

Join for Free!
119432 members


need help with subcloning !!

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

need help with subcloning !!

Postby Jasmine0507 » Wed Dec 21, 2011 1:20 pm

Hi

I am trying to clone a 350bp fragment from a pbabe into a plenti6 with N_terminal flag ha ( 6500bp). For this, I cut out the fragment with BamHI/EcoRI and I digest the plenti with BamHI/EcoRI as well. I obtain the fragment on gel and extract the band using machery_nagel gel exctraction kit, purify it. At the same time, I also obtain the digested plenti vector of the correct size. I ligate them using T4 DNA ligase (fermentas) overnight at 4 degrees and transform 100µl of the competent XL1 bacteria. For ligation, the vector : insert = 1:3 molar ratio and i take 100ng of vector and accordingly the insert.

Problem 1: I do get a lot of colonies on my main plates but a lot more on the vector only plates.

Problem 2 : however, i picked some colonies and did minipreps, did a Bam/Eco control digest to check if the ligation had worked. i get 6 out of 24 colonies positive i.e. I have the 350 bp fragment and the digested vector at 6500bp.I sequenced 4 of these 6 clones to do a final check if the fragment was correctly inserted. Now this is the point, where i am a bit lost!!

clone 1 : the desired fragment is present with the RE sites but there is a stop codon just after the flag ha tag in the correct ORF. I dont get it ??

clone2 and 3: the desired fragment is present in the correct ORF with the n-terminal flag ha tag but I cant find the site for ecorI, instead just after the stop TAG, there is GAATTTT in place of the usual GAATTC eco site. and this GAATTTT is actually present somewhere later in the 3'region of the plenti backbone, which means that my ligated vector is missing a part of the 3' region. The rest of the 3'backbone is fine. but the missing part had the blasticidn resistance gene, without which this plasmid is useless for me.

I would welcome any suggestions to improve my cloning strategy. Is there some star activity exhibited by EcoRI or plasmid recombination ?? How can I reduce the star activity of ecoRI if that is the case. Should I use some other bacteria or bacteria freshly made competent ?

Thanks a lot for your replies!!
Jas
Jasmine0507
Garter
Garter
 
Posts: 4
Joined: Thu Nov 26, 2009 2:19 pm

Postby JackBean » Wed Dec 21, 2011 1:29 pm

1) is there any reason, why bacteria with empty vector shouldn't grow?

2) are these real mutations or just the chromatogram is not good enough? Sometimes it looks as mutation on the chromatogram from sequencing, but closer look shows it is OK.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5693
Joined: Mon Sep 14, 2009 7:12 pm

Re: need help with subcloning !!

Postby Jasmine0507 » Wed Dec 21, 2011 4:55 pm

1) there is no reason why bacteria with empty vector shud not grow.. may be it just means that on my main plates as well i have more chance to have colonies with empty vector, which reduces the chances to get positive colonies. in one experiment i had 5-6 clones positive out of 24 , in the previous one none !

but I am wondering if my plasmid can self_ligate if it has been double digested ! (bam/eco) why do i have so low positive clones ? and if the eco digestion dint work properly, how do i still have the fragement inside teh strange vector that has lost some part.

2) the chromatogram is pretty good in terms of quality, no ambiguity ! I have checked again, I have Bam _ my fragment_stopcodon_ immediately after is GAATTTT no eco_site in 3' ??

:( i am not able to figure out the solution! the original plenti seems to be fine too, as other people are also using it.
Jasmine0507
Garter
Garter
 
Posts: 4
Joined: Thu Nov 26, 2009 2:19 pm


Postby JackBean » Wed Dec 21, 2011 6:08 pm

1) in such case it makes no sense to transform with empty vector. The only reason is positive control, which obviously works well ;)

2) then probably mutation during PCR or maybe some repair by bacteria after transformation
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5693
Joined: Mon Sep 14, 2009 7:12 pm


Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 0 guests

cron