I have been used taq PCR kit (1000u) so long time and may be I have used 10 kits (1000u) until these days. I have 3 kits already but when I start to doing PCR, I can get successful results with 10x PCR buffer but I can't succed with coralload buffer. I used it in many times when kits arrived me but I couldn't work with it. When I do PCR both 10x PCR buffer and 10x coralload PCR buffer, I can see good result with 10x PCR buffer but not with coralload PCR buffer. Now I have about 50 coralload PCR buffer tubes and I want to use it. I use same protocol when I do PCR work. I prepare pcr master mix according to kit hand book and put them on thermal cycler. After PCR reaction I prepare 1% agarose gel adding ethidium bromide and directly load PCR samples containing coralload PCR buffer. I can see how much my DNA go on gel but when I want to see my PCR result on uv transsimulator, I couldn't get any band untill today. I wonder, should I prepare my agarose gel without ethidium bromide? When I dying my PCR product that was done with 10x pcr buffer, my samples can dye with coralload buffer but when I use it alone my PCR reaction doesn't work properly. I mean, coralload PCR buffer is working as gel loading dye but on PCR reaction it doesn't work. I have no problem about product storege and other condition. Because all solutions (into kits) are work when use.
I wonder if coralload PCR buffer need specific PCR condition or imaging process or etc. I wonder your response immediately because I'm continue doing pcr, so I want to use coralload PCR buffer to easy application.
are you sure that you are using coralload buffer which contains MG and not coralload concentrate which is just buffer and coloured dyes. Pcr needs the MG to work and the concentrate is just for adding to the reaction mix after pcr to enable loading of the sample.
One other way of making buffers/reagents not work is by not mixing the reagent thoroughly after thawing. With glycerol in the mixture the solution thaws unevenly and the dense salts and glycerol stay at the bottom of the tube and water stays at the top. Taking material from top or bottom of the tube changes the composition of the buffer unless it is well mixed
Dear Paul,
Thank you for your response. I used MG contained coralload buffer (catalog number is: 201205) and also the buffer contain 15mM MgCl2 that was given in kit catalog. Also in some reaction, I used 25mM MgCl2 for PCR reaction. When I look at the qiagen taq DNA polymerase kit, the manufacturer is given same reaction condition and same mixture condition for 10x PCR Buffer and 10x Coralload Buffer. I use all solution with vortexing. When I buy this kit, I used it and I couldnt get good result with coralload buffer. The kits are stored according to the kits handbook and all product is working except for coralload buffer.
In that case your choices are
1 throw out the buffer that does not work. You have a working system so just use it
2 use the buffer just as a loading buffer and add at the end of the pcr to help the sample loading.
3 try using both buffers added initially...the coralloid will hopefully just act as a loading buffer and not interfere with the pcr.
4 Try lower annealing temperatures and optimise the coralloid buffer pcr from scratch
Do not worry about your gel. If you can see bands with size standard and ordinary buffer then the gel is fine.
Generally if I had a reagent that does not work I would throw it out
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