I had performed restriction digestion of insert and vector for 3 hours and gel extracted them using a qiagen gel extraction kit in 60 ul of nuclease free water. Thereafter I tried to measure the concentration using eppendorff mini spectrophotometer using 4ul sample+196ul enzmye but got strange readings (negative values) so I ran a gel using 10ul of the dilution made 1ul of extracted DNA and 5ul of extracted DNA. Below is given picture of my gel where
M-marker stepup 1kb ladder
A-vector 3.6kb 10ul diluted (50times)
B original restricted digested 1ul vector
C original restricted digested 5ul vector
D- Insert-1 1.5kb 10ul diluted (50times)
E-Insert-1 1.5kb 1ul original (50times)
F- Insert-1 1.5kb 5ul original (50times)
G- Insert-2 1.5kb 10ul diluted (50times)
H-Insert-2 1.5kb 1ul original (50times)
I- Insert-2 1.5kb 5ul original (50times)
As I am not able to measure exact the concentration of my restriction digestion samples of vector insert, how do I setup the ligation reaction. Any ideas or suggestions would be highly appreciated.
It has to be digested with NdeI and this enzyme is reported for exonuclease activity incubating more than 4 hours. plus enzymes were fast digest so the process is completed in 5-15 minutes as the manufacturer mentions but i had done it for 3 hours.
In addition to the above suggestions, I would suggest that you elute the DNA in max 30 ul, then I don't think it is necessary to measure the conc if you use the Qiagen Kit. then try to use 1:3 (2ul:6ul) or 1:2 ratio plasmid to insert in 10 ul total volume.
Good luck
To determine the units enzyme mass-time ratios, look to the manufacturer's unit definition. For example, if the enzyme has 1site per mole unit time on manufacturers template and you have 2, then you must double time or double enzyme or halve the enzyme.
Hi,
I would suggest you to use quantification of DNA by picogreen dye. The kit is available from Lifetech. It is highly sensitive dye which can detect even picograms of dsDNA.
Else, make dilutions in a quartz plate/quartz cuvette or any other UV compatible cuvette (dilution volume to be analysed should be ~300ul) make dilutions as 1:250, 1:500 and 1:1000 and take 260/280 ratio, once you get the readings, multiply them by the dilution factor, take the average OD and calculate concentration using following formula:
1 OD= 50ug/ml, so concentration= 50 X Avge OD.
Using nanodrop is not usually preferred for measuring DNA concentrations for reactions like ligation because these reactions are very sensitive for DNA concentration fluctuations. Nanodrop is useful when you want to know the concentrations approximately, like maxipreps.
Also, one suggestion, you should waste your precious gel-eluted sample by loading it in different volumes.
Last, from your gel pic, your inserts don't seem to be 1.5kb. Just a doubt!
Hope this helps. Keep us posted.
Good Luck... :)
One can concentrated restricted DNA by adding 0.3M sodium acetate final concentration and 2.5 volumes of 100% ethanol, followed by washing with 70% ethanol twice . Finally dissolving pellet in minimal volume of say 15 uL distilled water. Estimate concentration again on nanodrop. It is good to go for ligation reaction.
Hi, I agree with Saad, I always used qiagen gel extraction kit and I elute following the datasheet in 50ul or in 30-40ul if I want a more concentrated sample. After the eluition I run the plasmid, the insert (about 1-3ul, without diluition) and a marker with approximate quantification ( for example Fermentas gene ruler http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-dna-ladder-250-to-10000-bp/), I never used spectrophotometer to quantify digested products.
I think that you diluite to much the digested products, try 30 or 40ul of nuclease free, wait always 1 minute and then centrifuge. It is really useful to have a marker with approximate quantification, and calculate the molar ratio for your ligation. If you do not have this kind of marker you can, for example, measure few ul of your vector (3,6kb) from a maxiprep, with nanodrop, and run together with your digested vector. I always use an approximate quantification for ligation without problems.
before setting up ligation reaction, it's better to check your ligase enzyme, if you are sure about , start setting up ligase reaction based on answers.
Don't be too obsessed about having the exact DNA concentrations. You can easily estimate from your gels that all 3 DNA concentrations are pretty close with the vector being slightly less than the inserts. If I were doing it, I would take 5ul of vector and 5 ul of each insert, ligate and transform. Ligations are normally very forgiving if the DNA ends are intact and the enzyme is good.
Thank you all i followed ligation reactions and got colonies at 16 degree overnght incubation. the colony PCR is right as well as Restriction digestion also gave the same results. sent the plasmid construct for sequencing.
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