[CLONING] denatured fragments before ligation

Wolfgang Schechinger hubahopp at gmx.de
Wed Jul 21 10:41:19 EST 2004


</kernel panic> ;-)

Dear Fernando, 

would you give us a short description of what you actually plan to do, please!

(source of DNA, RE-enzymes used, strategy of cloning or whatever you want
to do)

please be as specific as possible.

Best regards, 

Wolfgang


At 08:22 21.07.2004 -0700, you wrote:
>Hi cloners,
>i'm working hard under the sun but trouble don't leave me alone...
>I've electroeluted with a dialisis membrane some fragments of DNA for
>a simple recombination step. Sizes are 7.2 and 1.2kb. Eluted in TAE,
>tot volumes between 1 and 2 ml. Agarose gel check of this is ok (yeald
>about 60-70%), one band per lane, no smear.
>Go on with a Microcon 100 to concentrate and to change the buffer. 2
>filters per fragment, centrifuged in batch of 10' till dry. Over the
>filter i can see fuxia colors due to EtBr. Wash and resuspend in 200
>ul TE.
>Thus phenol extraction, surnatant totally transparent (EtBr in the
>organic phase), ethanol precipitation ON -20=B0C, today morning i go
>checking and ... TADAAAA! Vector and one of the inserts with 2 bands!!
>One at the correct level, the other is neither plasmid nor
>contamination from electroelution (of the band left behind). My boss
>suggested they are ssDNA: i went to check the acqueous buffer storage
>phase of phenol -> pH 11! Checked pH of the purified DNAs -> 7-8. Thus
>we checked the reaction buffer of Ligase, 40mM TrisHCl pH 7.8 and we
>decided to correct the purified DNAs solutions to this value. Checked
>the pH, ok.
>Now enjoy this part ;-) in a thermal cycler i created a rinaturation
>program: 5' 95=B0C, 94=B0C 1', every next minute 0.3=B0 degree down to 42=
>=B0C
>(calculated to run for 3 hours). After 20' of high temperature we (me
>and the boss) realize that such high T aren't so much good, isn't it?
>Moreover the pH is 8... Stop it and reprogram for a scaling down from
>70=B0 to 42=B0. I do checks at half run and at the end: a big long smear
>from the 7.2 and 1.2 kb down, for all fragments... kernel panic!
>AYEAH!
>
>Now our RE seller will be happy to know that i have to restart... What
>can i change after the electroelution? Phenol extraction and
>precipitation first and then Microcon (to eliminate small impurities)?
>Maybe is it worth to electroelute with a small slice of agar and to a
>small final volume?
>
>Thanks a lot! Good work!
>
>
Wolfgang Schechinger


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