Protocol Online logo
Top : Forum Archives: : Molecular Cloning

got colonies but can't check them... - help!!! conference in 2 weeks!! (Jun/30/2006 )

ok that's it when you have conference or something everything stops working! mad.gif ive got clones....i didnt do negative control cause didnt have enough I check by PCR failure, I run positive control: failure ....(maybe because primers have different restriction enzyme sites attached but same cDNA dry.gif ) and i do miniprep (just sol1, 2,3) and do RE check and i can see faint band of my linearized vector but no insert... ohmy.gif ...maybe because too little of DNA (i have used all that I had)....what do I do?....i have a feeling that my dirty miniprep caused the problem but i can't just extract 20 colonies with phenol/chloroform... huh.gif ..and here they wont buy any columns... mad.gif ...what should I do??? any ideas for fast but reliable miniprep?? like with 24 well plates or something.... unsure.gif ....i can't repeat ligation....and even if i have the insert have just (or not) time to express the protein.... sad.gif


kathy, I know it's hard but you gotta slow down and focus on the incremental steps and not the presentation when you're at the bench, you'll go crazy and nothing at all will work for you. take a deep breath and take the experiments one step at a time and do the best you can.

just set up your cultures and do the minipreps. after soln 3, I find that an ISOH precipation followed by a 70% EtOH wash is the fastest way to get clean DNA for a digestion. for every 1.5 mL of turbid culture you prep, you should be able to resuspend in 50ul tris (TE, whatever) and cut 10-20 and see loads of DNA on the gel. If you were a little impatient and your cultures were wimpy, what I usually do in that situation is spin twice in one tube...add 1.5mL in the eppy, spin, dump the spnt, then add another 1.5mL and re-spin. (not necessary to increase buffers... basically it's the same as you would be doing if you waited till your 5mL overnight had heavier growth)

another place to save a little time is when drying the pellets. after the EtOH wash at the end, dry the tubes quicky in a temp block or a dry incubator with the lid open (has to be pretty clean, but as long as you're not keeping it around forever it works fine) for example, I'll decant the spnt and put the open eppy tubes in a temp block on my bench, next to a bunsen burner to keep most of the aersols moving up and not into the tube, set the temp at ~50 and they will dry pretty quickly.
don't let them sit there in the block after drying; I don't know what happens if you leave them too long but I bet it's not pretty

that's about the best I can do to help you shave time off the minipreps. anyone else have any tips?


I know your PI can be a jerk, but have you told him/her you just can't hit the deadline? it's best to do so NOW, while you can still come up with something else to present. last minute changes in abstract presentation is way better than no data and Kathy in the nuthouse

I hope I'm not coming off poorly; I honestly wish for you to do well and I hate to see you stressing out so much. I am not trying to be harsh or bossy. I do think you're painting yourself into a corner and I am worried it will get worse unsure.gif


sorry for late reply.
I do quite clean minipreps by sol 1 2 3, and then supernatant in 1ml 100%ethanol.
Then incubate 30' in 10mM Tris pH 8 + Rnase.
I use it directly for restriction analysis iwhtout any phenol chlo.
(and i put directly the whole digestion(20┬Ál + loading buffer) on gel)


I hope my response will be helpful although it may be late.

I normally do the colony PCR detecting the insert on the vector.

Transfer a single colony (just touch the colony you don't need the whole colony) using pippette tips to 20ul of water in 1.5 ml tube and boil (100C) in heating block for 5 minute. Put on ice to cool and centrifuge at 13000rpm on desktop centrifuge for 5 minutes. Use 8.5ul of supernatant in 25ul or 50ul PCR reaction with insert specific primers.