Course title
CT121W/CT122WPre-requisite
Biology or Honors Bioscience: Introduction to Biotechnology.Course description
This course is designed to develop higher levels of lab research technology currently used in the field of biotechnology. Students will develop laboratory skills; critical thinking; & communication skills currently used in the industry. Areas of study include cell structure; function & processes; molecular genetics; & microbiology. This course will prepare students for Advanced Biotechnology. This course fulfills graduation requirements.
Lab Example:
Name:
Date:
Manipulating DNA and Genetic Engineering: Restriction Digests of DNA
How can we take genes from one organism and connect them with genes from another organism?
Why would we want to do this?
Restriction digest plan
I?m using the restriction enzyme(s): HindIII EcoRI
I?m using the buffer color/number: yellow/2 blue/3
Step 1: Label your tube(s) with your initials and which enzyme will be in which tube.
Step 2: Pipette 10µl water into the bottom of your tube.
Now you will visit 3 stations. Please check them off as you visit each station so you don?t have to remember where you?ve already been. You can visit them in any order.
DNA Station Done
Add 6 microliters (µl) of DNA to your tube.
Buffer Station (Why do we add buffer?) Done
Which buffer(s) are you using? Add 2 microliters (µl) of buffer to your tube.
Enzyme Station Done
Which enzyme(s) are you using? Add 2 microliters (µl) of restriction enzyme to your tube.
Why do we use different buffers with different enzymes?
When you?ve visited all the stations; put your tube(s) into the 37∞C heat block or water bath. Why do we put the reactions at 37∞C?
Analyzing Your Restriction Digest Data
Materials
DNA from restriction enzyme digests (BE SURE TO ADD 5 MICROLITERS LOADING DYE!!)
agarose
Tris-acetate/EDTA solution (TAE)
micropipette/tips
electrophoresis apparatus
Procedure:
1. Get your electrophoresis apparatus. Make sure the comb is in place (there should be one comb near the black electrode) and that there are stoppers at both ends of the gel space.
2. Pour hot agarose into the gel space until it reaches the top of the gel box. Let the agarose harden; which should take about 10 minutes. Don?t touch/move your gel until it?s hard. Why not?
3. When the agarose gel is hard; take out the comb and stoppers.
4. Load your RESTRICTION DIGEST SAMPLES WITH LOADING DYE into the wells near the BLACK ELECTRODE. Why near the black electrode? Be sure to keep track of which samples you loaded in which lanes.
5. After you have added your samples pour TAE solution over your gel so that is it completely covered plus a little more. What do you think the TAE solution is for?
6. Run that gel!! Plug the electrodes into your gel box (red to red; black to black); being careful not to bump your gel too much. Plug the power source set at 100 V into an outlet. How can you tell your gel is running?
Draw a picture of your gel and label which samples are where before you add DNA to the gel.
After about 30 minutes the DNA should be sufficiently separated to analyze; the purple dye will have migrated approximately 2/3 of the gel; turn off the power and carefully remove the gel. The gel is very fragile; take care to not break it. You can remove the tray that you poured agarose on to and gently slide the gel into the staining tray. At this point you cannot see the DNA; what can you see and how do the different lanes compare?
Once you have placed your gel into the staining tray bring it to the staining station. Completely cover the gel with methylene blue and cover try with saran wrap. Stain overnight.
Next day--Viewing the gel: Pour the methylene blue back into the bottle and carefully place the gel onto a white light box. The gel is very fragile so take care to not break it. Draw a picture of your stained gel; be as accurate as possible in drawing the bands:
I used restriction enzyme:
How many cuts did your restriction enzyme make?
Using semi-log graphing paper; plot the distance a band traveled from the wells for the HindIII cut on the X-axis and the number of base pairs for that band on the Y-axis. With this information you can determine that sizes of the bands for the DNA cut with EcoRI. Use the table below to record your values. What is the relationship of the distance traveled for DNA compared to different sized pieces of DNA; remember you are using semi-log paper.
Restriction Digests of DNA
Table 6.1
Hind III
Actual Base Pairing (Bp) Sequence
Measured Distance (mm)
23;130
9;614
6;557
4;361 (generally very faint)
2;322
2;027
570 (may not be detected)
125 (may not be detected)
Table 6.2
EcoRI
Measured Distance (mm) Interpolated Bp Sequence Actual Bp Sequence
Band 1
Band 2
Band 3
Band 4
Band 5
Band 6
Band 7
Band 8
School Country
United StatesSchool state
ArizonaSchool city
GilbertSchool Address
2076 S. Higley RoadSchool zip code
85295Date submitted
Approved
YesApproved competency code
- CTE
- Career and technical education
- LADV
- Advanced science
- LBIO
- Biology