useful tips for low melting temperature gel purification. - please share your experiences. (Dec/09/2006 )
I love this technique, however i have many problems with it and at the least it becomes difficult to carry on some times. these are some difficulties i encounter:
1-1% low melt temp agarose breaks no matter what you do, and it is broken into halves before you manage to cut out the band. now i handle it on the parafilm but still sometimes it breaks.
2-following my protocol (molecular cloning by sambrook, i have to add 5 times LMT buffer to the gel piece to melt it. as i am not that good in cutting sometimes i end up with 400 ul gel piece. that means i have to add 2000ml LMT. that makes it impossible to put it in the ependorf and deviding between two tubes i risk losing DNA.
3-I precipitate with 2 volumes ethanol and 0.2 volumes ammonium acetate, it also gives me a volume which i can't handle in one ependorf tube.
4-after all this mess i end up with some small piece of agarose that i suppose contains my DNA as confirmed later by gel. it is fine i can dissolve it in TE buffer, but i am afraid that remaining agarose interferes with my cloning .
please tell me how do you solve those problems. thank you so much!
umm.... i use more then one eppendorf
I don't use low melting agarose... just plain old agarose. So the gel is nice and tough! I have found no significant difference between the two gel types, for DNA fragment below 10kb.
Well when faced with soft gels, my lab uses a large glass plate to move the gel around. Basically once running is over, the gel is moved to onto a glass plate. With the gel slab sitting on said plate, glass plate is lowered EtBr bath... stained. Glass plate is raised, UV, band excised on glass plate. YOu get the picture.
An alternative applicable to horrizontal gel boxes, is not to remove the gel from the gel caster/gel plate. And just do all the gel manipulation while said gel remains in its plate
And I use gel purification columns! (lucky me) and thus neatly avoid the agarose contamination problem. If you are worried about agarose contamination... be at ease as
1- you can remove agarose with B-agarase... it used to be a common lab enzyme until gel purification columns took over. The protocols must stills be floating about the next somewhere
2- there actually are protocol that call for DNA fragments to be ligated while embeded in low density agarose. So ligation does appear to be inhibited by agarose.
I found this protocol quite simple and less expensive than LMT agarose:
After running a gel with regular agarose. Cut the band and trim it to minimal size to get rid of the parts that do not contain your DNA band. Always use a handheld UV light source and set it to long wave length selection. Place the gel pieces in a tube top filter unit for 1.5 ml Eppendorf tube. Freeze it at -80 C till it become solid. Then take it out , place it in an microtube and spin it at top speed for 5 min. Add 1/20 vol 5 M NaCl, Phenol extract once and ppt it with ETOH at -20 C.
If you do not have a filter unit, make one yourself: Cut a 1 ml pipette tip from about 2 cm from the top using a razor blade into a tip and a top part. Further cut a ~0.5 cm long ring from the narrow part of the top part. Place the ring at the bottom of a 1.5 ml tube with narrow part facing up. Place the tip part of the pipet tip on the top of the ring. This provides a caution so that the tip is kept away from the bottom of the tube. Put some glass fiber at the end of the tip. Load the gel piece to tip and do the same as what's described above.