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BAC miniprep-high nanodrop value but NO BAND

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#1 agal



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Posted 29 April 2012 - 09:07 PM

HI All

I just started doing BAC cloning and things are not going well for me.
I have a 180kb BAC clone on a plasmid ordered from CHORI.
the thing is, minipreping NEVER works for me.
I've tried various protocols, mostly qiagen P1/2/3 buffers based, but also tried pheno extraction too.
here is a summary of what i do

pick fresh clone from plate, dip into 5ml LB/12.5 ug/ml cm
12-18 hours of shaking at 250 rpm
resuspend 250 p1 (qiagen buffer, ice cold, with RNASE)
add 250 p2, sit @ RT for 5 mins
(if P1 has lyse blue I always get blue clear result)
add 250 p3, sit on ice for 5 mins (ive tried 5-10 mins, in some instances 10 mins seemd to increase the yield, but not always)
(always get the white fluffy material)
spin 15000 rpm 15 mins (ALWAYS get a nice chunck of debri)
transfer supernatant
add 800ul isopropanol (room temp), mix, spin again 15000rpm 15 mins
(usually I will see a small white pellet)
remove iso, add 500ul 70% etoh, spin again for 5 mins
remove etoh, spin again for 1 mins, to collect all the residual etoh
remove trace etoh
air dry, i make sure the pellet turns transparent
resuspend in 50 ul H2O. I usually let it sit o/n at RT, but have tried sit 1 hour RT, or sit o/n in cold room. doesnt change the result.

from the above, i spec it on the nanodrop. the results are ALWAYS ranging from 800-2000 ng/ul.
in short, I have A LOT of DNA.

then I run it on a gel. even if I run 10ug according to the nanodrop (~10ul, usually), it shows up to be a band that has an intensity far less then one 1kb marker (which is about ~100ng).
which means the nanodrop is overestimating about 100fold.
the band size is correct (i guess) since it barely moves, (the size of my plasmid is 180k)
if i digest it (have to use a lot of my sample, ~40ul) the pattern is usually correct, but the bands are super faint.

I'm very sure that i always see a nice pellet after isopropanol. doesnt that mean I got a lot of DNA? then why am I having this problem?
any ideas?

Thanks in advance!!!!!


#2 Rsm


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Posted 30 April 2012 - 06:28 AM

I guess most of your DNA is bacterial DNA. This will stick to the well, and not migrate in a gel. You'll see only the (in comparison the bacterial DNA) low amount of BAC.
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#3 agal



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Posted 30 April 2012 - 10:44 AM

thanks for the input. that makes sense. What can I do to remove that?
right now I'm spinning at 15000rpm/15 mins to pellet down the cell wall/debri, shouldnt bacterial DNA come down at that point?
or should I increase my spinning time?

thanks a lot!

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