[CLONING] denatured fragments before ligation

Ferdinando Pucci brotizolam at libero.it
Wed Jul 21 10:22:57 EST 2004


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°C, 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°C, 94°C 1', every next minute 0.3° degree down to 42°C
(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° to 42°. 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!



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