Biotechnology Lab Bio 11 Week 1

Brief Overview of Lab Objectives Obtain Bacterial DNA (plasmids-pAMP and pKAN) Cut DNA into specific pieces using special enzymes (restriction enzymes- BamHI; HindIII) Measure size of pieces cut by enzymes (gel electrophoresis) Glue pieces together using other enzymes (DNA ligase) Take glued pieces and put them into another bacterium (plasmid transformation of E. coli) Separate bacteria with plasmid from those without

Today’s Objectives Obtain Bacterial DNA (plasmids-pAMP and pKAN) Cut DNA into specific pieces using special enzymes (restriction enzymes- BamHI; HindIII) Extract eukaryotic genomic DNA from strawberries* * Optional (Time permitting)

Prerequisites to lab Pipette tutorial (second) Metric system review (first)

Metric conversions 500µL = ______mL .1mL = ________µL 10mL= ________µL

Micropipettors Are fragile Expensive Precise They depend on correct usage for accuracy

Figure 5a: P-20 Model 6.86 m l = 0.00686 or 6.86 x 10-3 ml   Figure 5b: P-200 Model 132.4 m l = 0.1324 or 1.324 x 10-1 ml Figure 5c: P-1000 Model 262 m l = 0.262 or 2.62 x 10-1 ml

Steps in proper pipette usage Adjust volume Select tip Depress plunger to first stop Put tip into desired liquid Release plunger slowly Put tip into desired container Depress plunger to second stop Remove tip from container Discard empty used tip Repeat

Lab Concepts in Detail

Two Types of DNA in E. coli Chromosomal DNA – necessary for cell survival; circular, double-stranded Plasmid DNA – extrachromosomal DNA (“bonus material”) useful for experimental manipulation; circular, double-stranded

Plasmids contain nonessential (but important) genes “Bonus Package” 1: origin of replication and cloning site

Plasmids can be cut with restriction enzymes Enzymes homodimerize to make symmetrical cuts C G G A T C C A G C C T A G G T BamHI CG GCCTAG GATCCA GT “sticky ends”

Restriction Enzymes cut very specific sequences of DNA

Plasmid DNA manipula-tion is at the heart of biotech-nology Bacterium Cell containing gene of interest Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome Plasmid put into bacterial cell Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of interest Protein expressed by gene of interest Copies of gene Protein harvested Basic research and various applications Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

BamHI BamHI kanR pKAN pAMP HindIII ampR HindIII Ori Ori Restriction digest Ori BamHI Ori ampR BamHI HindIII HindIII (2332 bp) (3755 bp) kanR BamHI HindIII Ligation (784 bp) (1875 bp) BamHI kanR HindIII ampR Ori