BIOL. 101 Lab 7 Instructions and Notes

pdf

School

College of Southern Nevada *

*We aren’t endorsed by this school

Course

101

Subject

Biology

Date

Feb 20, 2024

Type

pdf

Pages

Uploaded by CommodoreElement12104

1 Lab 7 - Gel Electrophoresis Agarose Gel Electrophoresis (“running a gel”) Upon completion of a successful PCR reaction, each PCR tube will have billions of copies of the short region of the DNA we amplified with our primers (referred to as a PCR amplicon ). To visualize this amplicon we will use agarose gel electrophoresis . This technique is used to separate and view fragments of DNA based on size. Agarose gel electrophoresis can demonstrate whether or not DNA is present in a particular sample, and it can allow us to determine the size of the DNA fragments that are present. DNA has a net negative charge due to the phosphate groups in its sugar phosphate backbone. As a result, in an electrical field, DNA will migrate toward the positive pole. Agarose is a gelatinous medium that restricts the movements of large DNA molecules more than small DNA molecules. Smaller DNA molecules will move faster than larger molecules, and will migrate further into the gel. To perform agarose gel electrophoresis, each PCR sample is loaded into a well formed in the gel at the top of each lane. By including a set of DNA molecules of known size ( DNA ladder ) in one lane, the size of the PCR products can be estimated in the other lanes. The size of your PCR amplicon can be measured in the number of base pairs (or ‘ bp ’) in the PCR amplicon. Recall that base pairs are the A’s and T’s, and G’s and C’s that make the genetic code. Base pairs can be converted to kilobase pairs (abbreviated ‘ kb ,’ for kilobases ) using the following conversion: 1000 base pairs (bp) = 1 kilobase (kb) Answer some questions on this in the Workbook To perform electrophoresis, first you need to prepare an agarose gel. Your samples already have a gel loading dye added in from the PCR reaction. Gel loading dye performs two functions. First, loading dye contains sucrose, which will increase the density of your PCR product, allowing the sample to sink into the wells of the gel. Second, loading dye contains colored dye, allowing you to visually monitor the progress of your gel electrophoresis experiment. This loading dye was added into the primer mix from the last lab so is already in your samples. But this dye does not stain DNA; we also need to use a DNA stain called GelGreen to do that. Your instructor will add this stain into the sections agarose before teams pour their gels Part 1 - Preparing a 1% agarose gel Your instructor has prepared a 1% solution of agarose in TAE buffer. TAE buffer is composed of 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA, pH 8.3. The ions in the TAE buffer allow the solution to carry a current, which then allows the DNA fragments (PCR amplicons and the ladder fragments) to be separated according to size. To prepare the agarose gel solution, your instructor weighed out 1 gram of agarose for every 100 mL of TAE buffer (1 gram agarose/100 mL buffer = 1% solution). The solution was then heated in a microwave until the agarose powder went into solution. The gel has been kept at

2 55ºC to make sure it didn’t solidify or set. Follow the procedure below to complete the process of preparing a 1% agarose gel. Check box Preparing a 1% Agarose Gel Steps in procedure What is happening? Your instructor will demonstrate how to set up a gel tray. Each gel system is different so make sure you use the appropriate gel tray for the gel chamber you will be using Your instructor will add 20uL of GelGreen DNA stain to the sections flask of 400mL agarose solution What does the GelGreen do? As a team set up your gel tray and comb Why do we need to use a comb? Each team will pour in melted agarose until the gel is ~0.5 cm thick (about halfway up the comb teeth). Use caution – the melted agarose is 55ºC (or 131ºF) What time did you pour your gel? Allow the gel to solidify for 20-30 minutes What would happen if we pulled the comb out too early? While your gel is solidifying, practice how to load gels before you load your actual sample. Your instructor will demonstrate how to stabilize your hands when loading the gel. It is important to place the pipette tip just inside the top of the well, but not too far down because it can puncture the underside of the gel causing the sample to leak out ( Figure 3 ). It can be difficult to see the wells at certain angles so you may need to change your position to the gel. Practice loading wells using the plastic practice gels and the practice dye on your bench BEFORE attempting to load your samples. Cover the practice loading wells with water and make sure to rinse the wells after you use them. Everyone in the team should practice loading a well before moving on. Check box Loading Samples for Gel Electrophoresis Steps in procedure What is happening? Put on gloves. Clean all surfaces and pipettors with cleaner and paper towels. Why is it important to clean off the surfaces?

3 You will be sharing the power supply for your gel chamber with another team so move your chamber to that power supply now BEFORE you add liquid or load your gel. Carefully remove combs and sides from your gel and place the solidified gel and clear plastic gel tray in the gel box. Fill your gel tank with ~400 mL of 1X TAE buffer. The gel should just be submerged in 1X TAE with just a small amount of liquid right on top creating a smooth gel surface Why does the gel need to be submerged in TAE buffer? Obtain all the teams samples and the negative control from your instructor What were the labels of your samples? Thaw amplified PCR products which have been stored in the freezer (-20ºC). Flick and centrifuge them briefly (2s) Why did you flick and centrifuge the sample? Use the chart in the Workbook to record what samples will be loaded in what lane. Don’t forget to have a lane for the DNA ladder. Do this BEFORE you add the samples Add 5 μL of DNA ladder to the appropriate lane (your instructor will have the tube of DNA ladder for you to take from) What information does the ladder provide? Using a fresh pipette tip for every sample, add 5 μL of each PCR product (including the negative control) to its appropriate lane in the gel. When removing PCR product, be sure to put the pipette tip to the bottom of each tube and check that there is liquid in the tip when you remove it. Important - When you are finished, return your original PCR products to your instructor to be put back into the freezer. Why should you change pipette tips between each sample?

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

4 Once lanes are loaded, put the lid on the gel box, set it to 130V for 30 min or until the visual component of the gel dye gets close to the bottom of the gel. What do bubbles at the gel electrodes indicate? Part 2 - Gel Imaging The samples need to be visualized to see them and there are multiple ways to accomplish this. We used GelGreen DNA stain which will fluoresce under ultraviolet (UV) light. GelGreen is non- toxic, but you should still exercise caution when handling GelGreen or any gels in any lab. Avoid getting the gel or Gelgreen on your skin and wash your hands after using it. Choose one person in your team to handle the gel and this person must wear gloves at all times with the gel and not touch other things until the gloves are removed. Once our gels are finished running, we will photograph them using a Gel Imager: Check box Imaging Gels Steps in procedure What is happening? Make sure the blue filter is over the top of the white platform of the LED gel viewer box and slide the gel out of the plastic tray directly onto the blue filter Place the amber filter over the gel, so that the frame of the amber filter fits just inside the frame of the blue filter. Press the toggle switch on the front of the unit to “Blue.” Why do you need to place an amber filter over the blue light? The lights may need to be dimmed in the room to visualize it well. Take a photo with a team member's phone. If no team member has a phone capable of this, ask your instructor to image it Turn off the blue light before removing the amber filter. Place used gel in the gel disposal tray next to the viewer. Clean up the gel box, casting trays, and combs with tap water. Upload the image from the phone to the Lab 07 - Gel Image Template and then the Lab 7 Assignment in CANVAS with the file name “Lab 7 Raw Gel 1 Photo Your Team Name Date” ● This gel image is NOT your final image. It still needs to be annotated for full credit Why is this not your final image? Part 3 - Annotation and Interpretation of Your Gel Your gel image doesn’t mean much without labels. You need to annotate the gel with label s for ladder fragment size and what is in each lane. Upload the raw gel image into the template found here and follow the directions there to annotate it appropriately. Upload the annotated gel image to our Lab 07 Assignment in CANVAS with the file name “Lab 7 Annotated Gel 1 Photo Your Team Name Date”

5 ● Notes on interpretations of gels ○ It is common to see a diffuse (fuzzy) band that runs ahead of the 121-bp ladder. This is “primer dimer,” an artifact of the PCR that results from the primers overlapping one another and amplifying themselves. ○ DNA sequence can be obtained from any sample that gives an obvious band on the gel ○ If the negative control has a band then none of the team’s samples can be sent for sequencing

6 Lab 07 - Gel Electrophoresis Name:_________________________________________________________________ Team Name: _________________________________ Section #:_________ Workbook Questions Introduction ● If you had a PCR amplicon that was 739 bp in length, what would the length be in kb? ● What size PCR products do you expect to observe on your gel? (Hint: see your handout from last week!) Part 1 - Preparing a 1% agarose gel ● Why do we need to use a comb? ● What would happen if we pulled the comb out too early? ● Why is it important to clean off the surfaces? ● Why should you change pipette tips between each sample? ● What is the role of SYBR Green DNA stain in gel electrophoresis? ● Why does the gel need to be submerged in TAE buffer? ● What information does the ladder provide? ● What do bubbles at the gel electrodes indicate?

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

7 Lane # 1 2 3 4 5 6 7 8 Sample Part 2 - Gel Imaging Make sure to save your raw and annotated gel in the shared team google drive folder Lab 07 - Gel Image Template and download as a PDF and upload these files to the Lab 07 Assignment in CANVAS with the files names “Lab 7 Raw Gel 1 Photo Your Team Name Date” and “Lab 7 Annotated Gel 1 Photo Your Team Name Date” ● Who in the team uploaded the images? ● Brief written explanation of your gel electrophoresis results - which samples successfully amplified? Did your control amplify a product? What size (bp) were your amplicons? What size should your amplicons be? ● Brief Conclusions II: Does your team have any PCR amplicons that can be submitted for sequencing? If not, what are the next steps to get PCR amplicons that can be sequenced?

Related Documents

Week 4 Soap Note.pdf

NSG 6430 Week 9 Soap Note Meissner D.pdf

MCB2010L Practical 1 Study Guide completed.docx

Unknown Project.docx

BISC1112_Group_Activity_1_Spring_2024.docx

HLSS154FinalOutline.docx

Exercise 2 - Organ Systems Overview-1 (1).docx

NSG6435 Week 1 Soap Note Meissner D.docx

NSG 6435 Week 2 Soap Note Meissner D.docx

Exam 1_ TA Practice Questions (2).pdf

0 - intro to skeletal worksheet revised(1).docx

lymph histology worksheet.docx

Recommended textbooks for you

Human Heredity: Principles and Issues (MindTap Co...

Biology

ISBN:9781305251052

Author:Michael Cummings

Publisher:Cengage Learning

Biology: The Dynamic Science (MindTap Course List)

Biology

ISBN:9781305389892

Author:Peter J. Russell, Paul E. Hertz, Beverly McMillan

Publisher:Cengage Learning

Biochemistry

ISBN:9781305577206

Author:Reginald H. Garrett, Charles M. Grisham

Publisher:Cengage Learning

Biology 2e

Biology

ISBN:9781947172517

Author:Matthew Douglas, Jung Choi, Mary Ann Clark

Publisher:OpenStax

Biology Today and Tomorrow without Physiology (Mi...

Biology

ISBN:9781305117396

Author:Cecie Starr, Christine Evers, Lisa Starr

Publisher:Cengage Learning

Biology: The Unity and Diversity of Life (MindTap...

Biology

ISBN:9781337408332

Author:Cecie Starr, Ralph Taggart, Christine Evers, Lisa Starr

Publisher:Cengage Learning

SEE MORE TEXTBOOKS

Recommended textbooks for you

Human Heredity: Principles and Issues (MindTap Co...
Biology
ISBN:9781305251052
Author:Michael Cummings
Publisher:Cengage Learning
Biology: The Dynamic Science (MindTap Course List)
Biology
ISBN:9781305389892
Author:Peter J. Russell, Paul E. Hertz, Beverly McMillan
Publisher:Cengage Learning
Biochemistry
Biochemistry
ISBN:9781305577206
Author:Reginald H. Garrett, Charles M. Grisham
Publisher:Cengage Learning
Biology 2e
Biology
ISBN:9781947172517
Author:Matthew Douglas, Jung Choi, Mary Ann Clark
Publisher:OpenStax
Biology Today and Tomorrow without Physiology (Mi...
Biology
ISBN:9781305117396
Author:Cecie Starr, Christine Evers, Lisa Starr
Publisher:Cengage Learning
Biology: The Unity and Diversity of Life (MindTap...
Biology
ISBN:9781337408332
Author:Cecie Starr, Ralph Taggart, Christine Evers, Lisa Starr
Publisher:Cengage Learning