problem in cloning, if any one can help me out - (Oct/24/2008 )
Hi There,
I am trying to clone 3Kb fragment taken after RE digestion from pGL2 plasmid into another vector (4.5kb) having kanamycine as a selection marker called pEGFP-N2.
First time I cut pGL2 with smaI than with Eco47-III and finally with sca1 to distinguish between my desired band and the other one. the vector (pEDFP-N2) was digested with AseI, nhe1 and than Eco47-III, as my supervisor advised me to create blunt end so I did 5 prime overhang fill-in using Klenow polymerase after digestion with AseI. After RE digestion i run the samples on agarose gel, i got the bands one at 0.3kb and other one at 4.5kb for my vector (pEGFP-N2), as i was expecting and than i purified my desired 4.5kb band using gel DNA extraction kit. In case of pGL2 plasmid (that i amusing to take fragment of interest), i got four bands, first one at the position of 6kb (total size of pGL2 vector is 6kb, this is a light band that i think indicates that some of the DNA didn't cut, I used total 10ug of this plasmid ), second band was of 5kb(comparatively dark band), while third band was of 3kb (the fragment of my interest), and fourth fragment was of 1.6kb. I purified the band of my interest (3kb) suing the gel DNA extraction kit. I setup the ligation reaction in total volume of 40ul, using
3ul of vector DNA (4.5kb fragment of pEGFP-N2)
9ul of inserted DNA (3kb fragment of pGL2)
Water 1oul
10X T4 DNA ligase buffer 4ul (contain ATP and MgCl)
All of the above are added on ice
PEG 8000 (polyethylene Glycole) 5ul
T4 DNA polymerase 2ul
These two were added at room temperature
Ligation mixture was incubated at room temperature for 4 hours
Transformation was done using competent cells of DH5 alpha, the ratio of ligation mixture and competent cells was 1:5, first incubated on ice for 30minutes, than give heat shock at 42 degree C for 45 seconds; again keep on ice for 2 to 3 minutes and than added LB with out antibiotic and incubated at 37C for 1 hour.
After that this mixture was inoculated on LB plates having kanamycine and incubated at 37C for 16 to 18 hours
Butttttttttt, no colony was found next day, incubated for another 8 to 10hours but nothing found.
Than I tried to clone above mentioned 3kb fragment into another vector of 2.6kb, this time I did RE digestion using BamHI and HindIII for both of DNAs.t, his was a double digestion, than i digested my pGL2 plasmid with scaI to distinguish between my desired band and unwanted one.
Purified DNA from agarose gel using kit.
Did ligation for cohesive and in following manner.
Vector DNA = 2ul
Inserted DNA =2ul
Water 13 ul
Warm the solution at 45 degree C for 5minutes to melt any cohesive termini that reannealed, chill the mixture at 0C
Added 10x T4 DNA ligase buffer= 1ul
5mM ATP=1ul
T4 DNA ligase= 1ul
Incubated the reaction at 18C for 4 hours.
Transformation of same DH5 alpha cell was done asdiscribed above.
Againnnnn, didn't find any colony.
Can any one please help me out to get a successful cloning????
Cheers
Somi
While you have supplied us with a fair amount of information, there are some details missing.
Restriction: amount of DNA cut with how many Units of RE from which supplier, and for how long?
"3ul of vector DNA (4.5kb fragment of pEGFP-N2)" How many fmoles? "3 uL" doesn't give us molar ratios.
"9ul of inserted DNA (3kb fragment of pGL2)" How many fmoles?
Was the DNA exposed to the UV light for too long when you cut the band out of the gel?
Did you really ligate with T4 DNA polymerase or did you mean T4 DNA ligase? How many Units? What supplier?
5 uL of what concentration PEG?
Normally, the ratio of ligation reaction:cells is 1:20 to 1:50.
Using LB for expression rather than SOC reduces the number of colonies by 50%.
Restriction: amount of DNA cut with how many Units of RE from which supplier, and for how long?
"3ul of vector DNA (4.5kb fragment of pEGFP-N2)" How many fmoles? "3 uL" doesn't give us molar ratios.
"9ul of inserted DNA (3kb fragment of pGL2)" How many fmoles?
Was the DNA exposed to the UV light for too long when you cut the band out of the gel?
Did you really ligate with T4 DNA polymerase or did you mean T4 DNA ligase? How many Units? What supplier?
5 uL of what concentration PEG?
Normally, the ratio of ligation reaction:cells is 1:20 to 1:50.
Using LB for expression rather than SOC reduces the number of colonies by 50%.
First of all I would like to thanks for your reply, now come to the questions which you asked,
I did mostly over night digestion, using 5ul of DNA (2ug/ul, as my supervisor advised me to start with 10ug of DNA), 5ul of RE from NEB, 10ul of buffer and 80ul of autoclaved water= total volume was 100ul, I inactivated the first RE enzyme before using the second RE.
As far as molar ratio of vector and inserted DNA is concerned I don't exactly know but what I can tell you that I started RE digestion with 10ug/100ul for both of DNAs than I did 5 prime fill in using klenow polymerse and after 5 prime fill in I purified DNA using DNA purifying kit (only for vector), and I also purified the both of DNAs after agarose gel electrophoresis.
I am not exactly sure that it got exposed too long to UV light but I tried to cut as early as I could, however I will be more careful in future.
If I wroteT4 DNA polymerase sorry for that, it was T4 DNA ligase from NEB, 1ul of this in total 20ul of reaction mixture, using different concentration of vector and inserted DNA.
I made 40% of PEG 800 and than I used 5ul from it into 40ul of reaction mixture that provides 5% final concentration of PEG, I also tried 10% final concentration of PEG.
Could you please tell me that at what temperature PEG should be stored?
I hope I answer your required information. If you please help me to get a successful cloning I will be thankful.
Cheers
Somi