Hello All,
Many thanks to those who sent me replies. It seems that my problem was not
in the lysis, but the design of my oligos: With the addition of T7 RNA
polymerase initiation sequences, my primers perferentially amplified a
robust primer dimer. I have since succeeded in amplifying with shorter
oligos.
Based on the protocol I used, 1 =B5L of lysate (in SM) is fine for a 25 =B5L
PCR reaction. For a lambda gt11 phage plug eluted in 0.5 mL of SM, 1 =B5L
gave ~1000 plaques on RY1090, suggesting there are (at most) a thousand
dsDNA templates present at the start. The first denaturation step, 95=B0C
for 5 min, was sufficient to release phage DNA; the amount of product
recovered was not improved by boiling for five minutes prior to using as a
PCR template. The contribution of Mg++ in the SM (predicted to be about
0.3 mM) was not a problem.
Regards,
Morris
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>from Kouichi Iwasaki:
Hi Morris,
I have amplified an insert from Barstead cDNA library before. It has
worked well for me. I used 1 microL of the whole cDNA library, not a single
plaque. I used one primer to the Lambda vector and the other specific to my
gene. I found that during library construction, lots of artifacts occur,
such as small deletions at cloning sites. If your primers are very close to
the cloning site, there may not be that sequence in the vector any longer.
You could try another set of primers that are located further from the site.
Good luck on your experiment!
Kouichi
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>from David Baillie:
Morris,
How are [you] releasing the dna? I bet a mini protease K with
a 65=B0C digestion would do it. [Do] the ppt with 2 and half volumes
of 95% ethanol.
Dave
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>from Bruce Bamber:
Hi Morris
I did a bunch of that. One critical thing was to boil the supernatant for
a few minutes. I also had trouble with the 'official' primers AF77 and
AF78 so I made my own:
BB19 tgg agc ccg tca gta tcg gcg
BB20 gta gcg acc ggc gct cag ctg
finally, I used a funny pcr protocol originally suggested by V. Maricq
here's a excerpt from the unc-49 paper describing it.
(3) PCR reactions on lambda phage cDNA clones was performed as
follows: Phage supernatants were incubated for 5 min at
100=B0C. PCR with Taq polymerase was then performed as [usual]
except that an extension time of 7 min was used for 35 cycles. After
20 cycles of this program had been completed, an additional 0.5
=B5l of Taq polymerase was added to each reaction and the reaction was=
allowed to
proceed for the remaining 15 cycles.
good luck, and I hope you don't get too much wierd stuff, for which this
library is famous...
Bruce
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>from Michael Finney:
One possible problem: How much does the Mg from the SM buffer affect the
final concentration in the PCR reaction?
Mike
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>from Dattananda Chelur:
Hi
I have had no problems in using the lambda lysates directly for PCR.
Instead of using SM buffer, I use a buffer containing 2.5 mM
Tris-Cl(pH8.0), 2.5 mM MgCl2 and 0.01% gelatin for coring the lamda
plaque and I had used commercially available lambda gt11 forward and
reverse primers for PCR. I hape this pocedure will be helpful for you.
Good Luck!
Datta.
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>from Lei Xu:
Hi,
I have tried to do PCR directly from phage lysate. It worked very well.
What I did was to take a phage plaque and submerge it in 1ml SM buffer at
4 degree over night. Then I took 10ul of this lysate for PCR. The PCR
reaction is pretty much the same as normal PCR, except you need to adjust
the concentration of Mg2+. The [Mg2+] in SM buffer is around 8mM and I
usually adjust its final concentration to 2mM in the PCR reaction.
Hope this helps. Good luck!
Lei
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>from French A. Lewis, III:
Morris,
When I was screening Pete Okkema's library for a homologue to the
human FMR1 gene, I had done PCR from phage lysates. The way I got it to
work was to elute the plug in 1ml SM, then to amplify 20ul. I know that
it was probably overkill, but it is what worked. I hope this helps.
=46rench
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>from Mark Blaxter
Hi Morris
we do this routinely. (an EST project... 10,000 clones so far)
We pick the phage (just with a yellow tip) into 20 microl of SM, and leave
the tip in to diffuse for about 5 min.
We then take 2 microl of the phage to a 20 microl PCR (we use lambdazap, so
we can use M13 universal primers or T3 T7 etc)
The remaining 18 microl is mixed with DMSO to 5% and frozen at -80 for
later recovery.
Mark
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>from John T Jones:
Hello Morris,
We do this without too much bother. I'd heat your plug eluate to 95 for a
couple of minutes before using in a PCR reaction, or even just have a 2 min
at 95 step in front of your normal cycling parameters. The other thing I hav=
e
found is that you often need to do a dilution series, use 1 ul nite eluate, =
1
ul of a 1/10 diln and 1 ul of a 1/100 diln. Should dilute out any nasties
without ever making template DNA concn a problem.
Good luck!
John
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>from Andrew Papp:
Dear Morris,
Are you using EDTA or something else to make lambda
[release its DNA]? And then you may have to get rid of the EDTA
for PCR. I guess the cleanest would be SDS/EDTA, phenol extract,
and EtoH precip before PCR (?)
Andy Papp
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>from Robert Reis:
Morris,
As a former lambdologist, I'd suggest that you soak your lambda plaque
core in a dilute EDTA solution, NOT SM buffer (which tends to preserve
phage structure). In EDTA, lambda spews out its DNA contents intact,
and you are ready to PCR.
Robert J. Shmookler Reis
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[end of replies]
Morris Maduro
Department of MCD Biology
Biological Sciences II
UC Santa Barbara
Santa Barbara, CA 93106
lab: (805) 893-8090
fax: (805) 893-2005
