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DNA Sequence Questions summary

Harold G. Hills hhills at iastate.edu
Sat Apr 18 12:02:25 EST 1998


The questions were:

 How do you mix the loading buffer after adding it to the dried reaction
products before loading on a gel.

How do you denature, for how long, and do you ice the samples before load=
ing.


The short answer:  The more samples processed the less likely you are to
vortex or use a mixing device.  It is generally believed that once the
loading buffer contacts the pellet that the samples dissolve almost
instantly and only a brief mising with the pipet tip is necessary if at
all.   The key being getting the loading buffer to contact the pellet.

Most people denature in the 90-95=B0C range.  Some people ice and some do
not.  Does not seem to make much difference from the answers I got.  Some
use a lower temp and say the results are somewhat better.

The replies are listed randomly.

1. add loading buffer
  vortex tubes
 spin briefly
 heat for 5 minutes at 90oC  (we prefer 5 minutes instead of 2 minutes)
 put on ice until ready to load.

2.I just spin the dye down for about 15 seconds (no vortex,
but I ensure that the pellet side of the tube is outermost in the rotor  =
so
the dye contacts it well), heat for 2 mins (95 or 70C seem to work equall=
y
well) and put on ice till loading.

Incidentally, resuspended reactions retreived from a waste bin after 1 we=
ek
at room temp still give pretty good sequence also!

3. Once I have dried samples I add the Loading Buffer (4uL to dissolve
dRHOD reactions and 6uL to dissolve Big Dye Term. reactions).
The samples dissolve almost immediately.
Then I vortex for 5 seconds at least.
I heat the samples at 96C into a "Grant" block heater (not a water bath,
it is a metal-block).
Then I put the samples into a frozen aluminium-block and I store them at
-20C until loading.

4.I pipette up and down and scrape around the bottom of the tube with the=
 5
ul of loading buffer for about 5 sec.

One of our other techs adds the 5 ul loading buffer, vortexes briefly
then centrifuges briefly.

> We then heat at 95=3DB0C for two minutes and then put the tubes on ice =
until
> ready to load

5. 	1) We heat for 3 min at 90oC. The purpose of the heating is to
denature the DNA
fragments, so any temperature over 72oC should work reasonably well.

	2) After heating we store the samples on a Nalgene Laptop Cooler
(#5115-0032)
which has been stored at -20oC. The top is left off so the storage temp i=
s
closer to 0oC - I've had no problem with the sample freezing. The cooling=
 is to
slow down reannelling. I load the samples within an hour.

	3) Only one of my users uses the Amersham Kit. They ask me not to
heat the
samples at all. Results are usually good, but they do get compression blo=
bs in
their samples. I've tried heating to 80oC, but with no significant improv=
ement.

	4) After adding the loading buffer, we spin the Eppi tubes and
vortex them
before heating. Sometimes I'll vortex after heating. The sample is pipett=
ed up
and down before loading to further mix it.

6. I used to resuspend DNA in 1ul of loading buffer directly into the
96-well plates, vortex the plate for 30 seconds or so and let the
samples sit at room temp for a couple of minutes, then denature in
thermal cycler at 95C for 2 minutes and put in on ice immediately.

I have just come back from the Advanced Genome Sequence Analysis course
offered at Cold Spring Harbour and have learned that they don't vortex
the samples nor do they keep the samples on ice after denaturation until
loading.  I did not see much difference in the results and the DNA
certainly did not "renature" as ABI claims can happen if you don't put
the samples on ice immediately.

7.   We never vortex after resuspending
in loading buffer.  I tested it out and it made no difference.  We denatu=
re
either on a PE9600 followed by a rapid ramp to a 4C soak or on a 96-well =
VWR
heat block followed by quenching on ice.  We denature for 90C for 2 minut=
es
for sequencing samples and 95C for 3 minutes for genotyping samples.  I a=
m
not so sure that the ice quench is necessary.  Someone from ABI once told=
 me
it was not required.  I often let the samples warm up to room temperature
while loading the gel.

8.  we are working with 96 well plates here in Utah. After ethanol
precipitation, we resuspend the reactions in 2ul formamide dye mix, spin
the dye to the bottom of the wells at 1000rpm in a joun centrifuge with
microplate holders. We heat for 2 minutes at 70 degrees and place
immediately on ice. We load 1ul on 64 lane gels. This protocol works very
well for us. I think resuspension of the DNA is instantaneous as soon
as the formamide hits. We don't speed vac the DNA after ethanol ppt. If
you speed vac, you may have to resuspend for longer, but I don't think
shaking is necessary.

9.  we use Amersham loadiing dye where ever
possible now, it loads better than the formamide/dextran blue dye we used
to use.
The  96 well plates do not neccessarily need to be vortexed to mix dye wi=
th
dry sample before loading, but it can also be done very gently, the dye
doesn't go everywhere, and then we briefly spin the plate to pull sample
to the bottom of the well. We recently switched to precipitating and load=
ing
from the cycling plates and it seems to be working nicely. We heat the sa=
mple
to 90 C for a couple of minutes max. (if the samples have been frozen wit=
h
dye on them it will take the full 2 mins. samples can be frozen like this
for a couple of days with no deterioration, however reheating the same pl=
ate
with dye over again causes a loss of data quality)
the heating is done in an incubator/oven, with the samples covered to avo=
id
evaporation.
Finally we stopped putting our samples on ice a long time ago, I don't kn=
ow
if this increases the risk of compressions re-forming, but  50% of our
shotguns are terminators, and now with big-dyes this should be far less o=
f
a problem.

10.  I, too am in tubes for sequencing. I do not use Big Dyes, rather I u=
se
the PE/ABI Taq FS Dye terminator kits. I resuspend in the formamide:
TE(8.0) mix. I no longer bother to vortex. IMHO, the samples go into
solution quickly , almost instantly ( OH, I clean out excess  terminator
dyes  with Princeton sep columns because  of bosses orders....its his
money!) I heat  my speed-vaced samples  with the formamide/TE mix in PCR =
at
93-95*C for 2-4 minutes, then ice.( up to 7 minutes has not hurt the
reaction.......most times I quick spin the samples before loading...I kee=
p
samples on ice until I load ( within 1 hour of heating)
Pat
 PS. I do little plasmid sequencing, rather almost exclusively cycle
sequence PCR templates.

11. I hope you don't mind hearing from an industry type, but I thought my
two cents
would be useful.  Bottom line is I think there's alot of room to do what =
is
most
appropriate in your lab.

	1.)  Depending on how late in the day it is (and how tired I am), I
will vortex
anywhere from 5 seconds to 20 seconds/sample.  Yes I find it necessary to
vortex
in order to get as much of the sample as I can off the sides of the tube.=
  I've
noticed a definite increase in signal with vortexing ( or a least flickin=
g the
tube hard with your finger to get the formamide to splash on all sides of=
 the
tube).  After all, the volumes we're resuspending are pretty small.  I be=
lieve
the samples dissolve really quickly, it's just a matter of getting the so=
lution
in contact with the dried sample!  Some people here use the pipette tip t=
o
deliver the formamide, pipetting up and down while moving the tube around=
 to
mix, and it seems to work for them.(I've always wondered how much of the =
sample
remains on the tip).

	For 96 well trays, I usually invert the plate a number of times,
gently tapping
them on the lab bench to move the liquid around, and then spin them down.=
  You
can use a vortex, but I think the samples don't mix very well in those sm=
all
tubes.  However, the 3-8 =B5L or so you are using to resuspend in those t=
ubes
cover alot more surface area than in a 1.5 mL eppi tube, and so mixing do=
esn't
seem to be as much of an issue.

	2.)  I've always heated the samples at a high temperature...up to
~100 degrees
with no ill effects.  2 minutes will insure good denaturation, but I've h=
eated
them for up to 5 minutes (to concentrate a sample) with no problem.  You =
really
don't need to put them on ice.  The idea there was to snap cool them and =
"hold'
them in a denatured state.  I don't bother, and have kept them at room
temperture for  up to 15 minutes before loading without any problems.  I =
don't
know what temperature the Amersham protocol reccommends, but I've never t=
ried
anything lower than ~90 degrees.

	To heat the 96 well tray, we have a heat block with a 96 well
format.  I know we
got it from VWR, but I'm not sure who the manufacturer is.  I could look =
into
that if your interested.

12.  Make a mix of 1 ul EDTA:4 ul Formamide.  Add 4 ul to the dried pelle=
t.
Vortex very brifly. heat to 92C for 2 minutes, place on ice for about 1
minute.  Quickspin in a microfuge - just to get all the liquid into the
bottom of the tube.  Place on ice until ready to load -  within 10 minute=
s.

13. We have gone from tubes to 96 wells in the last six months, and are s=
till
working on exact details. When working in tubes, we did not find it
necessary to mix or vortex, but the techs would pipet the formamide mix u=
p
and down once or twice. If you hit the pellet, DNA should dissolve
instantly in the buffer. The only worry is possibly missing the pellet. I=
n
96 well format we use V bottom wells to collect the sample, and dry them =
in
those plates. The sample, as fas as we can tell from the hazy film in the
bottom of the well, can be quite concentrated in the V, or somewhat sprea=
d
out. So we do some mixing in the wells too.

	We have had sporadic problems with formamide producing ionic breakdown
products (formic acid), and producing curved gels. To address this proble=
m
we have gone from denaturing 2 min at 95, to lower temps, to not denaturi=
ng
at all. I think the formamide and the urea in the gel are sufficient to
keep denatured conditions. It is possible that compressions at the
beginning of the gel may resolve better with denaturation. At this time w=
e
load without heat denaturation, and we do keep the tubes or 96 well plate=
s
on ice. As we are exploring the formamide problem and modifying our initi=
al
deionization procedure, avoiding the heating and keeping samples on ice m=
ay
be less critical, but at this point we see a big difference in gel
curvature if do not keep samples chilled after resuspension. The occasion=
al
times when we have done 96 well format with heat denaturation, we have
tried collection into a PCR plate and denaturing in a cycler, and putting
the V bottom plate in a 85oC oven.

14. What we do is have everything in 96 well format fron the bacterial cu=
ltures
through loading the gel.  I just add 4 ul of the loading buffer with a mu=
lti
channel pipetor, shake the buffer down by swinging the plate a time or tw=
o.
Then I just hold it on the top of a vortex for a few seconds, make sure t=
he
sample is still at the bottom, then stick it into the thermal cycler and =
heat
it at 95 for two minutes, then stick the plate on ice until loading.  The
whole procedure takes maybe 5 minutes and works well in my hands.  Then I=
 load
with the hamilton 8 channel syringe which is a peice of cake.

15. We sometimes vortex and if not, manually wack the solution around ver=
y
well.  We also prefer to add the FE with the tubes in the speedvac so tha=
t
the pellet is definitely in the bottom of the tube and then spin the FE
down too to dissolve the pellet.

We use 95' and the original dyes were tested to be indifferent about mino=
r
temp/time variations.

16. We actually vortex, spin, vortex, spin, heat at 95C for 2 min, place =
on
ice, then spin again before loading.  We are still using tubes not 96-wel=
l
plates.

17. We work with sample sets of 64, in 96 well microtiter plates.  We hav=
e a
Genie vortexer, we hold the plate (in a microamp base) on the vortexer an=
d
vortex for 20-30 seconds.  We denature at 90 =B0C for 3 minutes in a PE 9=
600
or, on rare occasions, in a water bath at 90 =B0C for 2 minutes.  I had
spoken to Amersham about their lower anneal temps and they claim that the
lower temperature increases resolution.  I experimented once with pGem
controls/ABI rhodamine chemistry, denaturing at three temperatures and if=
 I
remember correctly, the lower temp gave nicer data.  Unfortunately, it wa=
s
at this time that the whole XL thing came to a head in our lab, I was
sidetracked, and I never had a chance to repeat or implement.  Thanks for
the reminder, I guess we'll look into this now.  We put the samples on ic=
e
immediately...we fill an empty container from Rainin p10 tips with ice an=
d
the microtiter plate fits nicely in this.  I guess the latest word, is th=
at
this step can be omited.

18.  When I was at ABI I resuspended the samples in formamide by pipettin=
g
up and down while stirring the bottom of the tube and never created more
work for myself by vortexing and then centrifuging.   Now
that I work for VGI which uses Amersham's thermosequenase kit, I too
noticed the lower denaturation time.  I suppose it works ok though I
find myself denaturing at 90 degrees just out of habit!  Just as it
takes an old dog longer to learn new tricks, it takes even longer to
unlearn old tricks!

19.  >I'm also interested in  how people doing 96 well format mix the sam=
ples.
I use 96 well plates all the time now. The pellets tend to resuspend very
quickly. I add my blue juice (using an eppendorf dispenser to put 4ul on
the side of each well then tap the plate to drop it all the the bottom) ,
denature and just pipette the sample up and down once before taking the
aliquot for loading.

I denature for 2 mins at 90C on a PCR machine then put the plate into a
small ice bucket thats got a slush in it so that all the wells are in
contact with icy water. If you put in onto plain ice the plate doesnt sit
flat.


>


Harold G. Hills, Ph.D. DNA Sequencing Specialist	515 294-9585
1184 Molecular Biology Building			    FAX 515 294-1597
Iowa State University                                   hhills at iastate.ed=
u
Ames, IA 50011-3260
http://www.biotech.iastate.edu/Facilities/DSSF/ABRF   For ABRF 97 DNA
sequencing tutorial.
http://www.biotech.iastate.edu/Facilities/DSSF   For info about facility.





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