Question

Gel Electrophoresis lab assignment The following sheets will be used to demonstrate your knowledge of gel electrophoresis. First please complete the lecture assignment using the posted gel electrophoresis module. After you have completed this assignment you should be able to understand and complete this lab assignment. Please follow the procedures as described below 1) Use the "ldeal" gel picture on page 4 for the following measurements. Measure the distance using a metric ruler from the bottom of the well in the gel (as marked for the first three measurements) to the bottom of the DNA band. This distance is the migration of the DNA fragment through the gel after the electical current was applied to the gel and the DNA that was cut with the restriction enzyme was added to the gel. 2) Record the distance of each migration for each band in the Hindill lane (colum) in the table on page 3 of this assignment. Repeat the measurements until all bands are measured and the distance in millimeters is recorded in the table. Repeat this procedure for BamHI, EcoRI and no enzyme bands. Why is there only one band in the no enzyme lane (column)? 3) The graph on page 2 can be used to estimate the base-pairs for each of the restriction enzyme (see the following for a brief description of semi-log paper:http://en.wikipedia.org/wiki/Semilog graph) the migration of the Lambda DNA fragments after being "digested" with the Hindill restriction enzyme. To graph this data you will use the estimated base-pairs found in the last colum on page 3. Use the migration measurements of the six bands and the cooresponding base-pairs in the table (e-g 3 cm for the 23, 130 base-pairs) to create a "curve." This curve will be used to estimate the remaining base-pairs for the rest of the bands found in the "idea" gel and recorded in the table on page 3. So, for whatever distance in centimeters you have for any of the BandHI bands you take the migration and follow it up to the point on the graph where it intersects the curve (remember you need to graph the migrations from Hindill first to create the curve!). You will take all of the migration distances for BandHI and EcoRI bands and use the curve created with Hindill to "estimate" the base- pairs for these bands. This is what is called using a standard curve to estimate sizes in base-pairs. Use this technique until all of the bands have estimates (in base-pairs) and have the table completed Save the completed document and upload this laboratory assignment before the deadline. If you have questions I also can walk you through this assignment by phone or email. Good luck

	L= No restriction	B=Bam HI	E=EcoRI	H=Hind III
Band 1	27mm	31mm	29mm	29mm
Band 2	NA	34mm	41mm	37mm
Band 3	NA	41mm	46mm	43mm
Band 4	NA	43mm	49mm	52mm
Band 5	NA	46mm	57mm	71mm
Band 6	NA	NA	NA	76mm

Above is the actual measurements for the distance in mm. Please plug this in with the existing chart located above

Answer 1

2. With out digestion with any restriction enzyme lambda DNA is a single intact DNA molecule which will produce a single band corresponding to a single base pair value ( about 27,000 base pair)
ANCE MIGRATED IN 10.000 ● Hindi 5.000 e Bam HI Eco R1 No Enzyme 2.000 5o0 10 12 Migration in centimeters (cm)

Data Table Directions. Measure the distance (in millimeters) that each fragment traveled from the well and record it in the table. Estimate its size, in base pairs, by comparing its position to the Hind III ladder. Remember, some lanes will have fewer than 6 fragments largest fragmentLNo restriction B BamHI E EcoRI H Hind 11 enzyme-just Lambda DNA restriction digest restriction digest of restriction digest of of Lambda DNA Lambda DNA irst Lambda DNA estimated estimated estimated base-pairs in mm distance distance in mm distance estimated base-pairs in mm 29 41 46 49 57 base-pairs 23,130 bp 9,416 bp 6,557 bp 4,361 bp 2,322 bp 2,027 bp base-pairs in mm 27 27,000 23,130 29 band 1 band 2 band 3 band 4 band 5 band 6 34 41 43 46 13,000 7500 6500 5400 7,500 37 5,400 43 52 3,600 71 76 4,900