Laboratory 2 Experiments

1. Laboratory 2 Experiments Genetics Laboratory BL415, Spring 2014

2. Outline

3. Restriction Digest of Lambda DNA Introduction: l DNA and Restriction enzymes

4. Lambda DNA can exist as both linear and circular molecules. After the λ bacteriophage infects E. coli, but before it inserts its DNA into the bacterial chromosome, it forms a ring, or circularizes, by the base pairing of the complementary single-stranded tails that exist at its two ends. These single-stranded ends are called cohesive ends or COS sites. This circularization will result in a difference between the restriction fragments produced by linear λ DNA and circularized λ DNA, as in the ring form, the first and last fragments remain joined because there is no restriction site between them.

5. One of Next Prelab Questions. Diagram a gel of expected patterns of DNA bands with lane 1 containing DNA markers (lHindIII, lane 2 containing DNA digested with BamHI, lane 3 containing DNA digested with EcoRI, lane 4 digested with HindIII, lane 5 containing uncut DNA, and lane 6 containing DNA markers (1kb ladder). On the side of the gel, clearly label the – and + poles of the electrophoresis and on the top of the gel clearly label your lanes (MlHindIII, B, E, H, UN, and M1kb ladder).

6. Procedure: • Obtain an ice bucket with ice, the linear l DNA (0.1mg/ml), the enzymes and sterile dH2O. • Set up your reactions as follows: • Cut (digest) l DNA with EcoRI, BamHI or HindIII (one enzyme per reaction, no double- or triple-cut) at 37oC in a water-bath for 60 minutes. Also, incubate the uncut l DNA prepared as indicated in the table above. • To each tube, add 2ml of loading dye (glycerol, EDTA and Bromophenol blue dye), quick spin the tubes and store them at -20oC until next lab period.

7. Drosophila melanogaster Dihybrid Crosses (In all photos, males are on the right and females on the left) Drosophila wild-type Drosophila mutant: vestigial wing Drosophila mutant: ebony body Drosophila mutant: white eye

8. X: female determinants Autosomes: Male determinants Y: not male determinants but sperm maturation XX: Female XY: Male XO: Sterile male or Y

9. Procedure: Dihybrid crosses Manual (2) pages 4-8 and 11-12 Each pair of students will be setting up one dihybrid cross, the crosses involve autosomal genes and sex-linked genes (Roman numeral is chromosome number; I is the X chromosome and Dominant allele is capital letter (or +) while recessive allele is lower-case letter. Note that each fly has the homozygous mutations on one gene and is wild-type for the other gene 1. both autosomal, unlinked: ebony body (eb, III) x vestigial wing (vg, II) eb eb VG VG x EB EB vg vg (eb eb + + x + + vg vg) 2. one is sex-linked: ebony body (eb, II) x white eye (Xw, I) If white-eye male: Xw Y EB EB x X+ X+ eb eb If white-eye female: Xw Xw EB EB x X+ Y eb eb 3. one is sex-linked: vestigial wing (vg, II) x white eye (Xw I) If white-eye male: Xw Y VG VG x X+ X+ vg vg If white-eye female: Xw Xw VG VG x X+ Y vg vg Different groups will perform the crosses with the first parent male, or the second male to see whether there are any differences in the reciprocal crosses. Autosomal cross Diagram the expectations for the F1 in cross 1 and diagram the expectations for the F2 in that autosomal cross. Sex-linked crosses, progeny depend on which parent had the sex-linked recessive alleles. Diagram the expectations for the F1 in crosses 2 or 3, for white-eyed male. Diagram the expectations for the F2 Diagram expectations for the F2 in crosses 2 or 3 with white-eyed female. Diagram expectations for the F2 phenotypes

10. Xw Y VG VG xX+ X+ vg vg Xw Y EB EB x X+ X+ eb eb Xw Y EB EB x X+ X+ eb eb Xw Xw VG VG x X+ Y vg vg Xw Xw EB EB x X+ Y eb eb Xw Xw EB EB x X+ Y eb eb Xw Y VG VG xX+ X+ vg vg Xw Xw VG VG x X+ Y vg vg eb eb VG VG x EB EB vg vg eb eb VG VG x EB EB vg vg eb eb VG VG x EB EB vg vg eb eb VG VG x EB EB vg vg Instructor podium