Bio. 551 Rec. DNA

Dr. Michelle Mardahl

Exam II. Study Sheet 37.5 points:

Know the function and applications of:

Poly A RNA

Total RNA

oligo d(T)

Broad Host range Plasmid

Transformation

Triparental mating

Conjugation

Post segregational killing

Incompatibility groups

Rnase H

MMLV Reverse Transcriptase

5-methyl dCTP

Eco RI adaptors

T4 DNA ligasse

Xho I/Eco RI

Competence

Competent Cells

Chaotropic salt

Guanidium isocyanate

Proteinase K

Acid phenol

Chloroform/isoamyl

Isopropanol

DEPC

RACE

RPA

Lamba Zap

Transduction

Transfection

Electroporation

Hanahan Method

Ca-Chloride

Transformation

LiCl

Northern Blot

Strepavidin/Biotin

RT-PCR

Magnetic streptavidin beads

Subtractive cDNA library

Sodium Acetate

B-mercaptoethanol

RNAsin

Isolation/Quantification & determination of the quality RNA - what are the species of RNA, and features of high quality RNA

Production of cDNA/cDNA libraries

Transfection Prototols- Liposomal/ Ca/PO4/ DEAE-Dextran/Electroporation/Receptor Mediated/ Gene Gun/ transient vs. stable

What are the controls needed for transfections- applications of use

Features of Broad Range Host Plasmids/ Applications of use

Steps of Conjugation

Tn5 Mutagenesis

EZ:TN mutagenesis

Current anticancer drugs induce apoptosis via several mechanisms. You work in R &D of a pharmaceutical company that targets transcription factors that influence cell death or survival. One gene family, Bcl-2, inhibits apoptosis. You want to test the efficacy of a drug ST1-571 on suppression of Bcl-X expression. Outline and detail the steps required for 2 different approaches that will facilitate your efforts (only one method can involve RNA purification). You must include appropriate controls. (10 points)

You work for Stratagene and your job is to make custom cDNA libraries. Your customer sends you transgenic mice tissues on dry ice. You use the Stratagene RNA Isolation Kit to extract RNA from your homogenate. List the function of the components listed below.

Denaturing solution (including guanidium isocyanate)

2M Sodium acetate

Water saturated Phenol

Chloroform::isoamyl alcohol

Isopropanol

Once you resuspend your RNA in 100 µl of ___________________, you quantitate the amount by taking a spectrometric reading of a 1/100 dilution of RNA. Given that 1.0 A OD₂₆₀ of RNA = 40 µg/ml, calculate the yield and determine purity given (1 point)

OD₂₆₀ = 0.4 and OD _260/280 = 1.912

How much of your sample should you aliquot to run out 1 µg of total RNA on a gel?____________________(0.5 pt). Next you use the Poly(A) Quik mRNA Isolation Kit (also made by Stratagene, of course) to isolate polyA RNA on the basis of oligo dT binding to a column under ______ salt conditions so that the poly A RNA can be bound, washed and eluted from the column under ________salt conditions. (1 point) After completion, you take 1 µg sample of total RNA (calculated above) + 1 µg of poly A RNA to run a 1.0% denaturing agarose gel. Draw the expected results from both samples, labeling the expected bands by approximate size (4 points)

Now that you are satisfied that you have high quality mRNA to construct a cDNA library, draw a flow chart to explain how to construct a directional cDNA library including enzymes and reagents such as polymerases, dNTPs etc. (5 points)

List 5 precautions that are necessary for working with RNA (2.5 points)

a.

b.

c.

d..

e.

Explain with flow charts how either the Tn5 mutagenesis protocol or how the EZ:TN method can be used to mutagenize genomic DNA. (5 points)