GE 50 reactions 79760 Recording Equipment User Manual


 
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spaced, a compression artifact is indicated. Try using the dITP reaction
mixture or a formamide gel.
Bands in 2 or 3 lanes
1. Heterogeneous template DNA (2 bands) caused by spontaneous deletions
arising during M13 phage growth. Try control DNA and limit phage growth to
less than 6-8 hours.
2. Insufficient mixing of reaction mixtures.
3. The sequence may be prone to compression artifacts in the gel.
Compressions occur when the DNA (usually G-C rich) synthesized by the
DNA polymerase does not remain fully denatured during electrophoresis. Try
using the dITP reaction mixture, or a 30-40% formamide gel.
If problems persist please contact USB Technical Support for assistance at
(800) 321-9322 or techsupport
@usbweb.com in the United States. For your
authorized distributor and support staff outside the United States, contact your
local GE Healthcare office. Contact information is listed in the
back of this protocol booklet.
CONTROL DNA SEQUENCE
The control DNA included in the kit is from pUC18, a double-stranded circular
DNA of 2.7kb. A partial sequence of this DNA is given below (14).
(Universal cycle primer)
5'-G TTTTCCCAGT CACGACGTTG TA->
AACGCCAGGG TTTTCCCAGT CACGACGTTG TAAAACGACG GCCAGTGCCA
10 20 30 40 50
AGCTTGCATG CCTGCAGGTC GACTCTAGAG GATCCCCGGG TACCGAGCTC
60 70 80 90 100
GAATTCGTAA TCATGTCATA GCTGTTTCCT GTGTGAAATT GTTATCCGCT
<--CTTTAA CAATAGGCGA
110 120 130 140 150
CACAATTCCA CACAACATAC GAGCCGGAAG CATAAAGTGT AAAGCCTGGG
GTGTT-5'(Reverse cycle primer)
160 170 180 190 200
GTGCCTAATG AGTGAGCTAA CTCACATTAA TTGCGTTGCG CTCACTGCCC
210 220 230 240 250
GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT GAATCGGCCA
260 270 280 290 300
ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC
310 320 330 340 350
TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT
360 370 380 390 400
CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGC