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Dissolve 20-base primer (MW = 6600) in 1 ml water. Pipet 5 l into 495 l water; measure OD 260 = 1.5. 1 OD 260 unit = 33 g/ml for single-stranded DNA. What.

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Presentation on theme: "Dissolve 20-base primer (MW = 6600) in 1 ml water. Pipet 5 l into 495 l water; measure OD 260 = 1.5. 1 OD 260 unit = 33 g/ml for single-stranded DNA. What."— Presentation transcript:

1 Dissolve 20-base primer (MW = 6600) in 1 ml water. Pipet 5 l into 495 l water; measure OD 260 = OD 260 unit = 33 g/ml for single-stranded DNA. What is concentration of undiluted primer stock? MW = 6600; 1M stock = 6600 g/l; 1 M stock = 6600 g/l = 6.6 g/ml 5 l into 495 l water = 1/100 dilution OD 260 of diluted stock = 1.5 concentrated stock = 1.5 OD 260 x 100 x 33 g/ml/ OD 260 = 4950 g/ml 4950 g/ml x 1 M/6.6 g/ml = 750 M

2 How much must you dilute concentrated primer stock to make 10 M solution? You have 305 M stock; want 10 M stock. Divide what you want by what you have: 10 M/750 M = = 1/75

3 20-base PCR primer = 50% G+C & 100% complementary to template; reaction contains 100 mM NaCl. What is T m of primer-template duplex? T m = 16.6 log [Na] (% G+C) /bp, where [Na] = molar salt concentration, % G+C = percentage as whole number (50% = 50), bp = length of DNA:DNA hybrid in base pairs. Tm = 16.6 log[0.1] x – 500/20 = 16.6 (-1) – 25 = 60.4 o C

4 Plate on ampicillin. tetA gene disrupted by lux operon. Plate untransformed cells as negative control. Ensures that competent cells were sensitive to ampicillin & tests whether medium contained sufficient ampicillin to kill untransformed cells.

5 Jargon:Preferred Term: a considerable number of many a decreased amount of less a majority of most a great deal of much absolutely essentialessential accounted for by the fact thatbecause count the number of count employuse

6 Jargon:Preferred Term: for the purpose of for is defined asis is similar toresembles it has been shown that (omit) prior tobefore red in colorred the vast majority ofmost utilizeuse

7 Define IMRAD. Introduction, Methods, Results, and Discussion. What is wrong with this title? Action of Antibiotics on Bacteria It contains general rather than specific terms. Action of Streptomycin on Mycobacterium tuberculosis is better.

8 What is one rule for the materials and methods section of a paper? It must provide enough information to allow colleagues to repeat the experiments. Which voice is usually best, active or passive? Active voice. What tense should you use to state previously published findings? Present tense.

9 When you describe your work in a paper, what tense should you use? Past tense. Which sentence is best? There is another method that is gaining acceptance. Another method is gaining acceptance. The shorter sentence is best.

10 gno-to-bi-o-sis: The study of organisms or conditions that are free of germs or contaminants or to which a known germ or contaminant has been introduced for purposes of study. Derived word: gno to-bi-ot'ic, adj

11 Will primer:template duplex DNA be more stable in 10 mM NaCl or 100 mM NaCl? T m is higher in 100 mM NaCl. High salt neutralizes charge repulsion between phospate backbones of two DNA strands.

12 DNA synthesis 20-base primer; 50 nmol DNA as dry powder. Prepare 100 mM stock. What volume of buffer used to dissolve DNA? 500 l 50 nmol/500 l = 0.1 nmol/ l = 100 nmol/ml = 100 mol/l = 100 M

13 8/ P EcoRI BamHI SalI PstI 8 kb vector plasmid EcoRI BamHI PstI SalI EcoRI ORF

14 BamHI 2, 4 & 6-kb fragments from desired plasmid. Opposite orientation 2, 2 & 8-kb BamHI fragments. Any other enzyme same pattern for both orientations

15 EcoRI BamHI PstI SalI P desired orientation 2 kb 4 kb 6 kb

16 EcoRI BamHI PstI SalI P opposite orientation 2 kb 8 kb


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