Laboratory: Unit 3: agarose gels

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Laboratory Unit 3 agarose gels sequencing
template preparation (page 56) Lecture review
agarose gel electrophoresis In-Class Writing
practice exam (pages 135-150) Read pages
96,135-150 xlii, section E Next Class exam
(class 13) Due Class 14 flow chart 4 draft of
editorial on GMO crops (pages 96 xlii, section
E)
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DNA Preparation for Sequencing DNA must be
free of contaminants. Submit samples in dH2O
or Tris, not Tris/EDTA.
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DNA Preparation for Sequencing Remove
unincorporated dNTPs primers (Qiagen kit).
10 µL of DNA/reaction _at_ 50 ng/uL 500 ng of
amplicon in 10 mL
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DNA Preparation for Sequencing Estimate PCR
product concentration by agarose gel
electrophoresis. Compare to DNA Mass Ladder.
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DNA Preparation for Sequencing Difficult to
estimate PCR product concentration with
conventional spectrophotometer. Use nanodrop
spectrophotometer to estimate DNA concentration.
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Sequencing Primers Primers (10 µL/reaction) _at_ 10
µM 10 pmol/µL 61.6 ng/mL for 8-27F primer
(MW 6161)
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Low Mass Ladder (Invitrogen) 2 31 agarose gel
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2 31 agarose gel
ng 200 120 80 40 20 (10)
bp 2000 1200 800 400 200 (100)
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BamHI
3 kb
2 kb
5.5 kb
4.5 kb
HindIII
BamHI
HindIII
1.5 kb
3 kb
0.5 kb
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gt 1 solution EcoRI site _at_ coordinate 6/0
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EcoRI cuts 3.5-kb PstI fragment ? 2.5 1.0 kb
PstI sites 1 2.5 kb from EcoRI _at_
coordinates 2.5 5.0
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EcoRI
6/0
PstI
1
5
pMB311 6 kb
2
4
2.5
3
PstI
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3rd PstI site 0.5 kb from another PstI site
(coordinate 4.5)
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EcoRI
6/0
PstI
1
5
PstI
4.5
pMB311 6 kb
2
4
2.5
3
PstI
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• PstI cuts 3.8-kb SalI fragment
• 1.8, 0.5, 1.5 kb
• PstI cuts 2.2-kb SalI fragment
• ? 0.5 1.7 kb

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PstI _at_ coordinates 4.5 5.0 ? 0.5-kb PstI
fragment in 3.8 kb SalI fragment Place 3.8-kb
SalI fragment on map in both possible
orientations. Only one ? 1.7 0.5 kb PstI-SalI
fragments ? SalI sites _at_ coordinates 0.8 3.0
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EcoRI
6/0
SalI
0.8
PstI
1
5
PstI
4.5
pMB311 6 kb
2
4
2.5
3
PstI
SalI
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20-nucleotide primer (MW 6600) in 1 ml 5 ml
? 495 ml of water Absorbance _at_ 260 nm 0.61
1 OD260 unit 33 mg/ml for ssDNA What is
concentration of undiluted primer stock?
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MW 6600 ? 1M stock 6600 g/l 1 mM stock
6600 mg/l 6.6 mg/ml 5 ml ? 495 ml 1/100
dilution OD260 of diluted stock
0.61 concentrated stock 0.61 OD260 x 100 x 33
mg/ml/ OD260 2013 mg/ml 2013 mg/ml x 1 mM/6.6
mg/ml 305 mM
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How much must you dilute concentrated primer
stock to make 10 mM solution?
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Have 305 mM Want 10 mM Divide what you want
by what you have 10 mM/305 mM 0.0328
3.28/100
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29 nmol/290 ml 0.1 nmol/ml 100 mmol/l 100
mM
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25-nucleotide primer 50 GC 100
complementary to template PCR reaction 100 mM
NaCl What is melting temperature (Tm) of
duplex DNA between primer template?
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Tm 16.6 log Na 0.41 ( GC) 81.5 -
500/bp Na molar salt concentration GC
whole number (50 50) bp length of DNADNA
hybrid
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Tm 16.6 log0.1 0.41 x 50 81.5 500/25
16.6 (-1) 20.5 81.5 20
65.5oC
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From the results of this experiment, it was
indicated that blue dextran (blue band) had a
greater molecular weight than DNP-glycine (yellow
band), as it was eluted first. Blue dextran
(blue band) eluted first, indicating that it had
a greater molecular weight than DNP-glycine
(yellow band).
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The procedures on pages 27-34 of the lab book
were generally followed with the following three
deviations as listed below The procedures on
pages 27-34 of the lab book were followed with
three exceptions
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Sentence 1 From the results From this
experiment This experiment indicated
that Sentence 2 do not end the sentence
with below. (Day, page 193) A preposition is
a poor word to end a sentence with.
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Both the crude and purified PCR products were
determined to be 560 base pairs in length. The
crude and purified amplicons were 560 base pairs
long.
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It was estimated that the crude product was two
times brighter than the ladder. The crude
product produced a band twice as bright as the
ladder.
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Identification of the unknown environmental
organism was identified as being Escherichia
coli. The unknown organism isolated from the
environment was Escherichia coli.
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DNA was extracted as described for Agrobacterium
above. DNA was extracted as described
(reference).
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Ends of restriction fragments produced by PstI
cleavage contain four unpaired bases that can
hydrogen bond to the complimentary bases on the
end of another PstI fragment. Ends of PstI
restriction fragments contain four unpaired bases
that can form hydrogen bonds with the
complementary bases of another PstI fragment.
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Colonies that were yellow in color and white in
color were seen. The colonies were either white
or yellow.
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A possible explanation for this result could be
due to the fact that molecules larger then the
largest pore size of the gel cannot diffuse into
the gel pores. Molecules larger than the pores
are excluded from the gel.
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Proper uses of that and which. (Day, page
196) Which ? nonrestrictive Introduce a
nonessential clause. That introduces an
essential clause.
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CetB mutants, which are tolerant to colicin,
also have All are tolerant. CetB mutants
that are tolerant to colicin also have Some
are tolerant.
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Active versus passive voice (Day, 198-9) Active
voice is usually better. S. aureus produced
lactate. (active) Lactate was produced by S.
aureus. (passive)
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In methods, passive voice is often valid. (Day,
page 67) Mice were injected with
(passive) We injected mice with (active)
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In unit 3, you cultured unidentified bacteria,
isolated genomic DNA, used this DNA as template
for PCR.
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Authors of the paper on bacterial diversity in
Amazon isolated bacterial DNA directly from soil
without culturing bacteria. Why did they use
this approach?
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They did not attempt to grow bacteria from soil
in culture because 99 of these species do not
grow in culture using current methods.
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List 3 parameters that affect Tm of
primer-template duplex DNA. salt concentration
length GC content number of mismatched bases