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problems with ligation

problems with ligation - Molecular Cloning Forum

problems with ligation - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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  #1  
Old 08-11-2008, 06:40 AM
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Default problems with ligation



i am rying to ligate a 6.1kb SmaI and NcoI fragment in a 9 kb pCambia vector0380 but with no success , anybody ready to help.
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  #2  
Old 08-11-2008, 07:32 AM
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Default Re: problems with ligation

hey scientistI,
do you have any results from your plates? did you get any growth on your negative control plates
those are quite big insert and vector how did you clone them/ligate them? What ratio did you use insert:vector?
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Old 08-11-2008, 10:11 AM
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Default Re: problems with ligation

Hi,

try to dephosphorylate the vector, and use 1:3 molar ratio of vector:Insert, before add the ligase into your reaction mix, try to heat up vector and insert mix to 55.C for 5 minutes, then add the remaining ligation mix and enzyme.

Thiru,
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Old 10-14-2008, 10:01 AM
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Default Re: problems with ligation

try and help!
we have amplified 3.4kb (5')segment and 2.9kb (3.9') flanking segment of sC gene from the genomic dna of aspergillus niger.these sequences have been cloned in TA clonng vector. to the 2.9 kb fragment we have ligated a 1.1kb bar gene(with promoter) to give a total of 4kb sequence. to this the 3.4kb fragment is ligated to get 5'sC-bar- 3'sC.digestions and pcr's are all working but the transformation of protoplasts with this piece on linearization we do not get bar expressions.

is there any way we can check whether bar is functional, it was pcr amplifed from he parent plasmid and then ligated to the 3' end
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Old 10-15-2008, 11:46 PM
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Default Re: problems with ligation

try and help!
we have amplified 3.4kb (5')segment and 2.9kb (3.9') flanking segment of sC gene from the genomic dna of aspergillus niger.these sequences have been cloned in TA clonng vector. to the 2.9 kb fragment we have ligated a 1.1kb bar gene(with promoter) to give a total of 4kb sequence. to this the 3.4kb fragment is ligated to get 5'sC-bar- 3'sC.digestions and pcr's are all working but the transformation of protoplasts with this piece on linearization we do not get bar expressions.

is there any way we can check whether bar is functional, it was pcr amplifed from he parent plasmid and then ligated to the 3' end

thanx
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Old 01-21-2009, 08:00 AM
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Default Re: problems with ligation

I'm having the same problem too, trying to clone a 8kb insert into a 5.5kb vector. But what i'm tryig now is to flood my vector with insert to more than 3:1 ratio, & leaving the ligation overnight at 16deg or 4deg, instead of room temp. It would be good to check if the vector is stable anough to hold big inserts. I'm using a low-copy number vector which people have used to clone big inserts & transform into stbl2 cells, hoping to stabilise my clones. Hope it works.
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Old 01-22-2009, 10:19 AM
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Default Re: problems with ligation

All the best yozyo! though it din't work for me.
I have another problem, can anybody elaborate on the mechanism of Homologous recombination in fungal systems. i have made several transformations though i get random integration i am not able to home in.
Is it possible to get HR in a single event,ie by transferring the parent transformant on seletion plates.

thanx
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Old 05-05-2009, 06:19 PM
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Unhappy HELP! blunt-ended ligation


I need help with sorting through my current predicament. I have been trying to blunt-ligate my inserts for a week but nothing working, here are the specifics.
My inserts are about 400bp and using the pBluescript II KS+ vector, which is 3kb in size. I first linearized vector using blunt-ended RE digest (Hinc II) then gel purified, phenol/chlorof and Etoh/NaAcet. I run on gel and nanodrop quantification is about 43ng/ul.
My inserts have a T7 promoter (for in vitro transcription), are PCR amplified and were gel purified too. They range in quantity between 12ng/ul - 25ng/ul.
used a 3:1, Insert:vector ratio for the ligation rxns.

At the end of each extraction, i set up the following rxn for a one tube blunt-end ligation
Our protocol is as follows; combine cocktail/2 ligation rxns as follows

10X T4 DNA ligase buffer 3ul
linearized vector xul
dNTPs 0.6ul
T4 DNA polymerase 0.25ul
T4 DNA ligase 0.625ul
T4 DNA kinase 0.3ul
ddH2O xul
final volume 10ul

combine 5ul of cocktail and 10ul of insert + ddH2O. incubate overnight at 12-14oC.
Run 5ul of ligation on agarose.

RESULTS
no ligation worked. the no insert control had linearized vector fragment only and the rest had just insert and linear vector size fragments on the gel.

I have run out of ideas at this time, please help. What could i do differently with my protocol. I could use a TA cloning vector but boss wants me (grad student) to clone into pBluescript, with this we can then linearize for blunt ends again before in vitro transcription.
help!!!!!!
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  #9  
Old 06-29-2009, 09:46 AM
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Default Re: problems with ligation

Hi all
For several months i have been doing fungal transformation, the transformants have been selected for phosphinothricin resistance and selenate tolerance.The sC gene have been disrupted with bar gene and should get ppt and selenate resistance.The vector has been so designed that sC gene has been disrupted with bar ie there is 3kb on either side sequences to home into the sC locus.

I do get good no. of transformants but after a while they lose either of the trait ie either the become bar positive or sC positive not both. I realise that while designing the construct there is 16bp identical sequence on either side of the bar gene. so probably they are alligning and exising the bar gene.

is 16 bp sufficient to remove my sequence of interest? how do i overcome this problem to get a stale transformant.

Thanx
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Old 04-10-2011, 01:30 PM
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Default Re: problems with ligation

i am facing problem in ligation m getting an extra band in addition to vector and insert band ............pls someone help me to find out where i was go wrong
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