Mostly, the DNA concentration of the pcr product after gel isolation is 10-17ng/ul (there is no peak at 260nm, even I used Qiagen kit). I am sure that I cut the right band from the agarose gel and ligate it. To increase the probability, 10ul (around 60-70ng Dna) transformation volume is used, and I had no colonies (STBL3 cells were used). Should I increase pcr product or enhance gel isolation procedure to take off all product?
What can be your suggestions?
(one of my gel photo attached,13ng/ul was isolated from the band at 8kb,it is too low concentration for me.)
Thanks in advance.
10-17 ng/ul is too low!!!
1. four 50 ul digest reaction or PCR reaction is needed for your work.
2. run 200 ul in gel by using wide comb.
3. bond is isolated from gel and purified.
4. now, DNA concentration is identified.
Hi Muge
It would be appropriate to increase the amount of the product, if not possible by PCR optimization do more PCR samples. However there is no need to load 200 or more microleters in a well...it will be difficult...rather you pool the samples, precipiate them, redissolve in less volume and then load in a regular gel for isolation of DNA for cloning ...I also agree that the concentration used by you in ligations are less.....if you want to work with those concentrations then use electroporation or similar methos for high transformation efficiencies....
bye
Ajay
Just wanted to add that It seems from the image that you have a lot of product in the gel, so you might want to modify your gel extraction (not the PCR). In our kit, for example, for larger bands it helps to warm up the elution buffer and to incubate the elution buffer in your column before spinning down: http://www.zymoresearch.com/downloads/dl/file/id/34/d4001i.pdf
Hope that helps!
To make a large/long well that will let you load 100-200 uL of sample, take some scotch tape (magic tape) and fold it over the comb, then use a razor blade to remove excess tape so that you have the desired number of wells connected, insert into agar and when it cools and you pull it out you will have a long well.
Also, check the gel purification protocol for large fragments, with Sigma's kit you have to add isopropanol and heat the elution buffer,
Thank you for your suggestion,i already add isopropanol but i did'nt practise to heat EB,i am thinking of it. Actually,i changed my protocol to avoid sample loss during gel purification.And nowadays i am tring to optimize DPNI cut after pcr reaction in order to get rid of backbone. But now some bands occur during pcr and it has nearly 1kb in size.Further steps such as ligation can it be problem?
- Badges
- Science topic
- Similar topics
- Gels