not getting transformants - (Aug/20/2007 )
hi
i have been trying ligation in molar ratios
but i am not getting transformants
i am just making the concentration by loading 1ul dna sample on agarose gel
as i am thinking that the lowest possible visibllity of band in gel may be like 15-20 ng
basing on that i am going furether is this right or not
i am not going for spectrophotometric analysis for checking concentration of DNA
please help me wether the concentration will differ with size (length ) of the dna
i am not getting transformants
please help me
thank you
If other things are correct, then ratios and amounts are much less critical than people seem to think. Look for your problem in other places, such as the cutting of the DNA, contaminants, or UV exposure. Are you doing controls for transformation efficiency?
hi yes i am keeping controls for transformation efficiency
in controls i am getting colonies
digestion also seems to be fine
i don't know whether the problem with ligation or some thing else
please suggest me on what basis exactly i have to take for ligation
in terms of molar ratios how i have to calculate and take
i will be very thankful if i get some suggestions
thank you
hi yes i am keeping controls for transformation efficiency
in controls i am getting colonies
digestion also seems to be fine
i don't know whether the problem with ligation or some thing else
please suggest me on what basis exactly i have to take for ligation
in terms of molar ratios how i have to calculate and take
i will be very thankful if i get some suggestions
thank you
hi still i am facing the problem
i am not getting transformants
this time i am purifying after first enzyme( Nde1) digestion and going for second enzyme digestion
ligating 1:2 to 1:5 molar ratio
transformation with controls
as i said i am getting colonies in controls
but i am not getting transformants and not even a single colony
this time i have plated on LB plates with 30ug/ml concentration kannamycin
pleeeeeeeeeeeeease help me
What is the efficiency of your transformations (a number, not just "I got colonies"). It is easy to transform E. coli with isolated plasmid, and can be hard if the efficiency of transformation is low to clone ligated plasmids. Transform your cells with 1 ul of plasmid (I use pUC19, but others will do) at a concentration of 10 pg/ul. You should get hundreds of colonies. If you get none, that is your problem.
as Phage434 has rightly stated, ligation ratio are not that important. COuld you explain what you are ligating? Where does the insert come from... is it a PCR product? What restriction enzymes are you using? Do you gel purify your fragments? Do you dephos your vector? What are the conditions you are using for the dephosphorylation? Is your lab experiencing any problems with ligations? Both the T4 ligase and ligase buffer do not tolerate freeze thaw cycles well and go bad quickly.
A control you can do, is to run some of your ligationed DNA (just prior to transformation) onto a gel. Firstly this is to check you really do have DNA still in your tube (if you are using electroporation, the ethanol percipiation step can lead to lost of DNA), and secondly to check that the ligation has worked. AS you will see high molecular weight bands.. of ligated molecules.
hi
i have transformed only 1 ul of uncut plasmid for checking transformation efficiency 'and i got more than 100 colonies in a plate
the same with single cut and ligated plasmid as a contol that also gave more than 100 colonies in a plate
what may be furether problem
plaese suggest
thank you
A control you can do, is to run some of your ligationed DNA (just prior to transformation) onto a gel. Firstly this is to check you really do have DNA still in your tube (if you are using electroporation, the ethanol percipiation step can lead to lost of DNA), and secondly to check that the ligation has worked. AS you will see high molecular weight bands.. of ligated molecules.
*insert is 600 base pcr product
*restriction enzymes are BamH1 and Nde1
*gel purifying before going for ligation
Thus the question becomes, how many basepairs have you added around the NdeI site. The NdeI site requires at least 7bp (I prefer 8bp) of skirting on either end of the restriction site to enable the site to be cut at any efficiency.
Thus the question becomes, how many basepairs have you added around the NdeI site. The NdeI site requires at least 7bp (I prefer 8bp) of skirting on either end of the restriction site to enable the site to be cut at any efficiency.
yes as i have mentioned earlier there are no additional base pairs around the Nde1 site