Question

Chromosomal and plasmid DNA can be cut into manageable pieces by restriction enzymes. Using agarose gel electrophoresis, the DNA fragments can be separated on a gel, based on their lengths. In order to see the fragments, a stain is typically added to the gel. The size of each fragment can be determined by comparing each one to a DNA molecular weight marker of known size.

Below is a map of pBR22 plasmid. The position and base pair number of the recognitions sequence for different restriction enzymes is indicated. The fat arrows designate the position and size of several genes, for example, ampicillin and tetracycline resistance.

Using the attached plasmid restriction map answer the following questions.

1. What is the size of the pBR322 plasmid?
2. What is the size (in base pairs) of the ampicillin and tetracycline resistance genes?
3. How many DNA fragments would you get when digested with the following enzymes? What would their sizes be?
   1. EcoRI
   2. HincII
   3. BtgI
   4. EcoRI + HincII
   5. EcoRI + BtgI
4. If you were to run these digests on an agarose gel how would the gel look? (Draw a picture of the DNA fragments on the gel below). Blue boxes represent the wells that the digests would be loaded into. Label each well with what digest will be loaded into it. Don’t forget to include the marker!

Use the following as a marker (M)

Use the following as a marker (M) 10000

  

Post lab questions:

1. Agarose gels can be used to study what size of DNA fragments?
2. If agarose gel material is labeled as 1%, what does the 1% refer to?
3. What causes the molecules to be separated on an agarose gel?
4. Name two common DNA stains that are used to visualize DNA on an agarose gel?

RESTRICTION MAP: P.S. THERE IS NO MORE INFO. THIS IS ALL MY TEACHER GAVE US FOR INFO.

Answer 1

plast - What is the size of PBR 3 22 An 436l be oR 4.361 kb. - what is the size in base pairs) of ampicillin and tetracycline resistance genes? Aus Ampicillin - As ampicillin resistance gene, An flanked by And I (3365 be location) and Earl (4 155 be location). Thus, size of 4155 - 3365 2794 bp upprox. Tetracycline - A tete flanked by Ew Rv (185 bp locus) and PFIMI (locus-1315 bp). Thus, size of tetk is is 1315-1852 11 1315-1852 113o be approx. - How many DNA fragments would you get when digested me and what would be with the following enzyme as theis size? - The das wid enzyme recognition site in the plasmid are Hinell 3905 EcoRl 4359 XBgI 528 PBR322 X Btgl 1447

a) Eco R2 – A single-cut at 4352 bp loans 4359 linear DNA (частьr) DNA fragment al DNA size a 4361 be b) ninell - A single-cut at 3905 locus. linear DNA (4361 bp) DNA fragment al DNA fragment size 2436l be c) Big 3 - Double-cut one at 528 and other at 1447 , alabe 3442 528 34 42 be neuf- I hp Fragment - 2 1919 > 1447 No. of DNA fragment 22 DNA fragment size a fragment I 2 3442 bp ment 2 2 91969 02

d) EcoRI & Hinell - 435 454 bp 3905 3859be toringuent - 2 fangenen No. of DNA fragment 22 agments size a ol. 2 3859be 02. 2 454 lop e) EcoR2 + etga a 4359 91969 horagment ! 2. 528 530be 144+ fragmenta 2912 be ament 3. No. of DNA fragment 3 size of ONA fragment a al. 2919 be 02. 2530 be 03. a 2912 be 03

Magher ce EcoRL Minell Byl Ecofl ecoll ninch att L.wells i Тоооо 80 + 6000+ $oot 400O1 3ooof 250of 2004 1500t 1 voot 7507 500 + 250 oy.