Sounds like dye blobs ,
We usually get bad blobs when users use ethanol/salt precipitation instead
of isopropanol .
Here is our modified isopropanol precipitation protocol you can try.
PS Our original ABI 377 Black casting frames are getting quite old ,
springs have lost tension. We are wondering if we should switch to the new
grey casting cassettes any comments about the new frames? Are they better?
MODIFIED ISOPROPANOL PRECIPITATION PROTOCOL
BIG DYE ONLY! Please follow times listed as closely as possible.
This protocol works for both 1/2 and full reactions, do not halve the
volumes if you are using 1/2 reactions.
1) After your PCR reaction transfer your reaction mix to a NEW 1.5ml
eppendorf. USE AS LITTLE OIL AS POSSIBLE in your PCR's. There should be no
oil left in you sequencing samples as it interferes with gel loading and is
often associated with salt and dye blobs. Excess oil can be removed by
placing your reaction mix on a piece of parafilm then pipetting off the
reaction mix leaving the oil behind.
2) Add 120ul of 70% Isopropanol (2-propanol only), vortex and leave at room
temperature for 15 minutes. DO NOT LEAVE FOR MORE THAN 30 MINUTES as you
can start to precipitate unincorporated dye.
3) Centrifuge for 20-40 minutes at max speed.
4) Immediately aspirate off the supernatant . It is usually easier to
remove most of the liquid, respin for 10-20 sec then remove the remaining
liquid with a pippetor. There should be no visible liquid leftover and be
careful as the DNA pellet is soft and can easily be dislodged. Try to use a
pippette tip that has a very fine or bevelled point (e.g. gel loading
tips), especially for removing the last droplets .
5 ) Rinse the pellet with 500 ul 70% isopropanol, mix then re-spin for
20-40 minutes.
DO NOT LEAVE the pellet in isopropanol for long periods.
The pellet is usually very small and may not be visible .
If the pellet is large and coloured (pink) you should redisolve in 20ul
water and repeat the protocol as you probably have too much precipitated
dye and or salt.
6) Once again remove all liquid as completely as possible! Dry your samples
under vacuum or in a speedy-vac for a few minutes.
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