shorter vector fragment at the end of the cloning - (Dec/11/2006 )
Hi,
I am trying to clone 1,8 kb insert into 4,7 kb vector(pEGFP-N1). after performing the standard cloning protocols (digestion, quantitation,ligation, transformation,miniculture and screening w/ the restriction enzymes of plasmid isolates) I observed my insert as 1,8 kb on the gel but for my vector fragment a 0.9 kb of its part is missing i.e Its length was 3,8 kb. I am sure of the length of the insert and vector and running them together with DNA i got after restriction digestion. and see the vector fragment part is shorter. can anyone help me to figure out the problem pls.thank you
I have a question, is this truncation seen in all the clones that you have recovered? How many clones have you checked?
What enzymes did you cut your insert and vector with? Asumming the following senario was true ; some RE enzyme from your insert got into the ligation reaction and cleaved your vector: will you see a truncation of the vector?
yes actually I did two separate ligation experiment and screened 10 different colony miniculture in each totally 20. and i got the same pattern of bands in each which was short vector fragment.
What enzymes did you cut your insert and vector with? xhoI & bamhI for both vector and insert. the insert was obtained after designing restriction sites containing primers and pcr and topo cloning,mini culture w\ RE
Asumming the following senario was true ; some RE enzyme from your insert got into the ligation reaction and cleaved your vector: will you see a truncation of the vector?
so you mean contamination of my insert. but i did gel extraction after digestion of topo vector. so does restriction enzyme run on the gel w\ insert?
no. But there are many ways you could have done this cloning. One of which was to digest your PCR product, phenol/chloroform and then directly clone your PCR product into the pEGFP-N1 vector. In which case, there is a small posibility that some restriction enzyme might have survived.
I would like to ask what strain of e coli are you using for your transfromation. Do you know its genetype. Is there any sequence similarity between your insert and vector.
Is it possible to find out the location of the truncation on your vector?
so you mean contamination of my insert. but i did gel extraction after digestion of topo vector. so does restriction enzyme run on the gel w\ insert?
no. But there are many ways you could have done this cloning. One of which was to digest your PCR product, phenol/chloroform and then directly clone your PCR product into the pEGFP-N1 vector. In which case, there is a small posibility that some restriction enzyme might have survived.
I would like to ask what strain of e coli are you using for your transfromation.
chemically competent TOP10 cells
Do you know its genetype.
from the manual of this competent cell product I can find it. but is it important
Is there any sequence similarity between your insert and vector. no i do not think so
Is it possible to find out the location of the truncation on your vector?
well i sent the cloned vector for sequencing. and got no sequencng results. so one primer was targeting the cmv promoter site and the other the n terminal of gfp seq. so the truncated part could either site i think
currently I am as puzzled as yourself. Do you know the history of your vector. THere might be the off chance that your vector has been manipulated in its past, and thus no longer exactly like pEGFP-N1.
THe only thing I can think off at this moment is to redo the transformation but grow your cells at 30 Celsius. Do all your cultures for cells with this plasmid at 30 Celsius. This is the voodoo answer whenever one meets a DNA structure which is "unstable"... for unknown reasons.
hi, i was also working with EGFP-N1 for some time and the problem was that I got no fluorescence after I cut out the insert (we received it from some poeple). 
Could our problems be related to some properties of this vector? 
This is a little out there but I have had problems with ligations that result in transformants carrying plasmids smaller than the start plasmid. I haven't done the experimentation but I have prevented this from happening by being very careful with exposure of the DNA to UV. My idea is that damaged DNA transformed into E. coli will result in the hosts repair system cutting out the damaged bits during replication resulting in a truncated plasmid. So, just be very careful with your exposure to your UV and run your DNA on the gel in the dark if you intend to cut it out (cover the tank with a box). This may not be your problem but its good practice.
Quite right.
UV exposure, even at long wave lenght settings should be measured in seconds. You have to work very fast and efficiently. Thus the horror stories of people getting sunburns using the transluminator.. means some users are way over exposuring their band to UV light