Determining optimal digestion time for partial digestion
Posted 18 October 2011 - 11:30 AM
Posted 29 October 2017 - 01:46 AM
Procedure to obtain homogenate of T. cruzi from epimastigote culture useful for coating wells MTP: taken 1 ml of epimastigote pure culture (forms cultured at 28 ºC in LIT liquid medium), and centrifuged in 5 ml Eppendorff tube at 4000 rpm at 4 ºC for 10 min. Discard the supernatant and 1 ml of phosphate buffer saline (PBS) at pH7.2 is added, and the tube vortexed. This washing operation is repeated for three times. Then, 1 ml of PBS was added and the tube vortexed. Aliquot and store at -20 °C.
Posted 20 July 2018 - 05:52 AM
You don't provide much information to go on, including the purpose of the partial digest. Usually it is to obtain a band for cloning, so I will assume that it the reason. In general you test several time points, run them on a gel (as you apparently have done), then look at the gel. You need to know what your size standards are, and what size your desired band is. Look at the various digests and pick the one that gives the most of the particular desired band size. Then repeat the most successful reaction for the time point that worked best. Don't scale up in the same tube-you don't want to change any of the conditions (including rxn volume). But you can do additional identical digests in the same size tubes for the same time. Then after you stop the reaction with heat or whatever, you can pool them (or not, depending), concentrate them and run on a gel to isolate the correct band. Whether you pool or not depends on how close the unwanted partially-digested bands are to the one you want. If they are real close, concentrating the reaction products could cause the bands to overlap (desired with undesired) and you won't be able to separate them. If they are far apart, you can concentrate and run the digest in one (or few) wells on the gel. If the bands are close together, run the rxns in separate wells on the gel, to prevent overlap. Finally excise the band you want and purify it from the agarose with a kit. Run a sample on a gel to see how sucessful yo were at getting rid of all the unwanted bands. This takes a little practice to get it right, but you learn from every mistake, and you will get better at it with time.
Edited by OldCloner, 20 July 2018 - 05:56 AM.