Eco47III is it problematic? - Selfligation doesnot work with Eco47III... (Dec/28/2006 )
I am in to deep trouble. I had cut my plasmid with the Eco47III and it had produced the linear band on the gel. After that I had added linker to it and ligated (also the positive control was set). But, to my surprise there was no colonies in (Vector+Insert and Vector). Then again I tried the same thing with the other plasmid having the single site of Eco47III. And set the self ligation for both the plasmids cut with Eco47III. But again after transformation there was no colonies.
Does anybody have faced the similar problem.
All the suggestions are welcome!!!
Hope to hear some of u...
The problem further increased...
I had cut the same vector with the SmaI and tried to religate it. But to my surprise got only 2 colonies instead of the loan. Can anybody tell me what could be the reason for the failure?
From all these thing I had concluded that there is some problem in my technique or handling as all the other people are using the same thing and getting the results.
Your advice/suggestions/help is dearly needed.
Well SmaI is a blunt end enzyme. And blunt end ligation is not the easiest things to do. What are you ligating conditions? How long, at what temperature and how much DNA and how concentrated is your ligation mixture?
look into your ligation box and conditions as suggested by perneseblue. check if other poeple are also involved in blunt end ligation and are using same ligation mix and same conditions as yours. also consider that your vector is not so pure, maybe prepare another miniprep.
smaI doesnt digest completely and overdigestion is a possibility with this enzyme. I would try to use some other enzyme for ur cloning. I dont use smaI when I plan to use the DNA for ligation purposes, only for digesting mini or maxi preps.
At last after many attempts I could succeed in doing blunt end self ligation.
Thanks to the 5x Blunt End Ligation Buffer (BRL). Now I will use the insert with it.
The problem is also with Eco47III enzyme as it is giving less colonies as compared to EcoRV.
Thank you very much to all for your valuable help.
I will keep you posted.
T4 DNA polymerase is a single polypeptide of 114 kD. The enzyme lacks a 5’ > 3’ exonuclease activity, so it is comparable to the Klenow Fragment, but T4 DNA polymerase has a much more active 3’ > 5’ exonuclease. P. O’Farrell describes the action of T4 DNA polymerase and Tris® Acetate Buffer (TAB), which is a universal restriction enzyme and ligation buffer. (P. O’Farrell 1981 BRL Focus 3 (3) 1-3; P. O’Farrell, 1980. MGG 179: 421; and 1981 BRL Focus 3 (4) 6 indicates that the unit definition in the original paper did not agree with BRL’s unit definition.)
T4 DNA polymerase is the product of the bacteriophage T4 gene 43. T4 DNA polymerase has 3’ exonuclease activity in the absence of dNTPs, and polymerase activity in the presence of dNTPs. The 3’ exonuclease or proofreading activity acts on all 3’ hydroxyl termini. Since the enzyme preferentially binds internal regions of DNA, it is very difficult to saturate the 3’ ends with enzyme. May be inhibited by steric structure of the DNA template, such as when having its polymerase activity activated by Gene 32 protein, at which time the 3’>5’ exonuclease activity is completely inhibited. (Amersham pharmacia catalog 1999, p. 109.)
The exonuclease activity is dependent on the enzyme:DNA ratio. The dependence is roughly linear at enzyme:DNA ratios below 6.25 units/µg of DNA (40 nucleotides/min/3’ end). At higher enzyme:DNA ratios, dependence is non-linear and reaches an apparent rate of 120 bases/min/3’ end at a ratio of 75 units/µg DNA.
The optimum pH is 8.0-9.0, with only 50% activity at pH 7.5 and 9.7. The optimum magnesium concentration is 6 mM. The enzyme also requires SH reagents for maximum activity. It is inhibited when the total ionic strength of the reaction mixture exceeds 100 nM.
In the presence of a single dNTP, the enzyme idles between exonuclease and polymerase activity. The addition of all 4 dNTPs will cause polymerization at up to 15,000 bases/min. In the absence of accessory proteins, secondary structure will impede the enzyme.
When labeling, the enzyme’s idling action removes a terminal 3’ dATP about 10 times faster than a terminal dCTP.
T4 DNAP can be used to fill in 5' overhangs to create blunt ends.
I assume that when you say "PCR purify," you are referring to a PCR clean up kit? While you have a lot of clean up steps, I have found that the cleaner the DNA is for a blunt end ligation, the more successful you will be.
Gel extraction of fragments greater than 1 kb should be performed immediately after running the gel. Some lots of agarose permanently trap large fragments if the gel is allowed to sit for an hour.
I recommend BRL's ligation buffer (which contains PEG) for blunt end ligations. 5x Blunt End Ligation Buffer (BRL Technical Bulletin 5224-1 1992.): consists of 250 mM Tris-HCl, pH 7.6, 50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% (w/v) PEG 8000. To prepare 10 mL of buffer, weigh 2.5 g of PEG 8000 in a 15 mL Falcon 2097 tube that has been treated with antistatic spray or wiped with a sheet of fabric softener. Microwave a 100 mL bottle of Type I water for 1 minute on High. Add 4.4 mL of hot water and 2.5 mL of room temperature 1 M Tris-HCl, pH 7.6 to the PEG and immediately mix to dissolve. Cool the mix to room temperature and add 500 µL of 1 M MgCl2, 500 µL of 100 mM ATP and 50 µL of 1 M DTT to a final volume of 10 mL. Aliquot for storage at -20°C. (If you do not use hot water, the PEG will require an overnight dissolving step.)
What is your insert:vector ratio? I recommend a 2:1 ratio for a 1.5 kb insert into what I assume is approximately a 3 kb vector.
So, after very long process of trial and errors I had reached to the conclusion that there are some problems with Eco47III (AfeI) as it is giving less colonies after self ligation as compared to the EcoRV for my plasmid.
5x Blunt End Ligation Buffer is the best thing for bluntend ligation. So, feel free to make it in ur lab and use it for your blunt end ligation. It works exceptionally well.
Today I am transforming my actual blunt end ligation.
The good news is that I got about 40 colonies. I picked up 18.
Lets see what happens next!!!
Thanks again for your efforts and interest in this topic.
I am using the approximately 1ug of DNA for the digestion with SmaI (3 hrs) and column purify, using 5ul of that (after diluting it in to 40ul of ddH2O). Using the T4 DNA lifase (Fermentas) with 10X ligation buffer @ 16C, overnight in total volume of 20ul.
I had prepared the fresh vector thrice and that too column purified. Used the gel extraction columns for the extraction.