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Dear Lynn, 
I just received the map of the pTURBO helper plasmid. Surprise! It's
pUchsDelta2-3. Maybe this deserves mention in the alias list for
delta2-3. Have you gotten any info on the size discrepancy between the
7.3KB on my map and the 8+ KB on your compilation?
Frank
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Subject: 
Dear Lynn, 
The following is a table of predicted fragment sizes from your
compilation, and the actual sizes of fragments that I obtained from a
given restriction enzyme. Please note that these are really crude
estimates for my diagnostic purposes. See if you can make any sense
out of these. If you need more accurate numbers, I can re-perform
these experiments with the same or different enzymes if you see
fit. Note also, that in estimating these fragment sizes, I assumed
that the plasmid was 7.3 Kb which was reasonable on the gel system I
used. Let me know if I can be of further assistance.
Frank
Predicted (bp)	Actual (Kb)
	BamHI
5392		4.8
2686		2.5
	Eco RI
5838		4.8
2240		2.5
	HindIII
5709		3.5
1531		3.0
830		0.8
	SalI*
4384		5.5
3676		1.8
18
	XbaI
6916		7.3
1162
\* The third fragment (18bp) would have been missed on the gel system I used.
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Sent: 	Friday, November 01, 1996  8:25  AM
Subject: 	Re: pUChsDelta2-3
Frank,
Thanks for the info.  Looks more screwed up than I had realized.
One quick question: did you run the BamHI and EcoRI cuts on the same
gel?  In other words, do we know for sure that both cuts produce the
same size fragments (whatever the actual size of those fragments
may be)?
I assume that you have used this thing and know that it works as
advertised...
--Lynn 
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Subject: RE: pUChsDelta2-3
The BamHI and EcoRI digests were run on the same gel in adjacent
lanes. Being that they were exactly the same size fragments, these
would be candidate enzymes to repeat in the event that you can't make
sense of these fragments. The plasmid described here has bestowed many
hardy transformants upon me. Keep in touch.
Frank
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Subject: puchspidelta2-3
Hi Lynn, 
I was able to do some more digests of the helper plasmid. The
following is the list of fragment sizes (actual) vs expected from the
compiled sequence:
Eco RV
Expected	Actual
8078		7400
Hind III
Expected	Actual
5709		3450
1531		3000
838		1000
Pst I
Expected	Actual
3890		4950
1511		1300
1212		900
797		760
668
Sst I
Expected	Actual
5283		5375
2795		2175
Xba I
Expected	Actual
6916		7400
1162
Xho I
Expected	Actual
7103		7400
975
Bam HI
Expected	Actual
5392		4750
2686		2600
Eco RI
Expected	Actual
5838		4700
2240		2600
Sal I
Expected	Actual
4384		5375
3676		1700
18
Pvu II
Expected	Actual
2364		2400
1856		1900
1595		1000
896		800
794		*one of the smaller must be a doublet
573
These sizes are approximations based on the results from DNA mobility
analysis software, but by "eye", they look reasonable. Since you
compiled the sequence, perhaps these numbers can give you an idea
where the problem resides. I know time is short at this point,
however, if you can find the problem with the sequence (or Map), and
you need additional digests, or double digests etc., to verify the new
compilation, let me know.
Frank
Frank J. Fokta IV
Department of Biology
Marquette University
Milwaukee, WI
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Sent: 	Thursday, March 06, 1997  2:07  PM
Subject: 	Re: puchspidelta2-3
Frank,
I've played around and get a good fit for your restriction data if I:
1) Reverse the orientation of the vector segment (thus reversing the
restriction sites at either end of it).
2) Replace the pBR322-Hsp70 fragment with 800 n's (no sequence,
800bp long). 
I did number 2 because none of the sites that should be in this
segment appear to be there: SalI, XhoI, XbaI, PstI.  I predict that 
there IS a PvuII site.  It really looks like this fragment is NOT
what it is advertised to be.  But.. it works...so what does that
mean ??!!!
May I cite our correspondence as a personal communication
to FlyBase?  I would like to attach some brief cautionary explanation
to the map. 
--Lynn
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Subject: RE: puchspidelta2-3
Hi Lynn, 
I have to agree...something is screwed up. I'm going to test another
source to see if the plasmid is the same (the third one!). I will also
try a couple of double digests this weekend. Maybe this will shed some
light on the situation. ... Yes, you may cite our correspondence. I'll
keep you updated.
Thanks, 
Frank
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