PTC Lab Prep for last day - can be done anytime in week before

Dilute Buffer as directed - TBE, if precipitated, heat and put on stir plate to get back in solution Need 1 L for gels and another 2L for gel boxes (2 if block periods and can share boxes, 3-4 if running 12 boxes simultaneously)

Note - I need to make my own gels - at least measure out the agarose (NOT agar) and make the solution - then give it to Richard to boil. Then I add the Gel Green. Then he can pour and do the combs etc.. Then test one of each batch with Marker DNA a day or two before hand to be sure they will work.

Make 2% gels - 2 g agarose per 100ml 1XTBE (50 ml per gel)

• Use the 15 lane combs since need 2 lanes per person, so each gel can hold ladder on each side and 6 students or one lane ladder and 7 students
• So will need 5-6 gels per class
• If doing EtBr, use Janet’s protocol at 50 ug per 500 ml
• If Gel green, use 3-5ug per gel

PTC Lab Prep

Before Day 1

• 10 ml Aliquots of Gatorade or 9g salt/1000ml distilled water - make a few extra
• Rehydrate Chelex - 15 ml dH20 into 1.5 g of Chelex
• Aliquot Chelex using p1000 set to 100ul - be sure to swirl before each aliquot so that you can see plenty of beads in bottom of each tube
• Print rosters, pin #’s for kids, with 4 columns for each day of lab: 5 pts per day and +1 for kids who carry through their partners when absent
• Students who will be absent - have them do spit and make first tube

​

On Day 1

• Set out heat block - set to 99 degrees at least 1 hour before class and push start - should see the blinking light next to heating
• Use large microcentrifuges set to 10,000 (can turn up to 14000 for kids whose pellet not forming well)
• See station set up from Carolina teacher prep
• Have a large rack numbered using tape for turning in DNA tube at end - DNA into freezer overnight
• Ice is not needed today
• Check that final DNA tube does not have Chelex beads

PTC Lab Prep ctd

Day 2 - Recommend just buy a couple bags of crushed ice for the next few days

• Have extra Dixie cups for absent students
• Crushed ice in styrofoam cup per group
• Have PCR tubes pulled apart and put in the crushed ice right after- emphasize to keep this cold
• Kids use special microcentrifuges (with PCR tube rack) to mix before turn in
• See station set up
• I defrost primer 15 min prior but keep it in white ice block - I dispense 22.5 ul to each and leave the tube in the white ice block as I pull up
• PCR program (PTC) - amount is 27.5ul (28ul)
• For turn-in blocks - keep blue blocks on crushed ice to keep frozen

​

ALSO - put out practice gels (in cabinet with practice dye), tupperware, dye, and practice paper with circles

PTC Lab Prep ctd

Day 3 - Consider having one table set up and rotating through 2 groups at a time?- then could keep PCR tubes in blue blocks

• For turn-in blocks - keep blue blocks on crushed ice to keep frozen and floats for uncut on crushed ice also
• Crushed ice in styrofoam cup per group - kids get PCR tubes and put into these. Have them come up as a group with ice cup to get enzyme
• Kids use microcentrifuge tubes to mix before turn in
• See station set up
• I defrost RE 15 min prior but keep it in white ice block - I dispense ONLY 1 ul to each and remember to use what’s in cap
• PCR program (Digest) - amount is 19 ul, 2 hours then 4 deg hold

Day 4

• See Station setup - set up 12 boxes so 6th and 7th can run simultaneously
• Can run odd/even periods on different days or on a block day ideally
• Keep marker in white ice block as it gets passed around
• Run at 160V for approx 45 minute (or 5 cm) - don’t set the time, and don’t pause or open them during run
• Draw orientation of post-it on board, wells on bottom/sticky end - write names sideways
• 10-15 ul into wells -

Notebook - 1.4.1, 1.4.2, 1.4.3 - 80 points

1.4.1 - 24 pts

1 - #1-3 research on how vaccines work (5)

2 - Causes of Death 1900 vs. 2000 (5)

3 - Top 10 Public Health Interventions (10)

4 - 9 CQ (9)

​

1.4.2 - 26 pts

5 - Making Vaccines Presentation Notes (4)

6 - 6 Different Types of Vax (6)

7 - #14 drawing and explained (5)

8 - 11 CQ (11)

​

1.4.3 - 30 pts

9 - Epidemiologist Career Journal (3)

10 - #5 Cholera research and #6 Steps that Snow took in the cholera investigation (5)

11 - Task 1: 5 questions (2)

12 - Task 2 Attack Rate (5)

13- Task 3 Epidemiologic studies (5)

14 - 10 CQ (10)

​

11/18 - Warm-up: Good Things

​

1) Introduce Socratic Seminar preparation - watch CRISPR video

2) Number notebooks for notebook check

Assemble case files and review order of presentations

• 2 printed copies of your written case report
• 3 printed copies of your patient materials (you keep 1 for your role play and 1 each for myself and guest)
• 2 rubrics with all of your group member’s names on each

​

3) Finish presentations

Homework -

• All 2.1.1 including CQ due tomorrow
• Review PCR with video 1 and 2
• 2 articles due Friday - 1 of them is scientific about CRISPR

​

​

Introduce Socratic Seminar preparation - watch CRISPR video

Unit 2 Key Assignment – Gene Therapy:

At the beginning of the unit, students will be introduced to the assignment of performing a review of current scientific literature on the most promising modes of gene therapy, especially CRISPR- related technology. Throughout the unit, they will be asked to find and read at least 4 articles (2 of which must be from scientific journals) and create an organizer summarizing the following for each article:

1. Description of the gene therapy procedure
2. Strengths/Advantages of the procedure
3. Weaknesses/Concerns of the procedure
4. Who is performing this procedure or will be likely to perform the procedure?
5. What types of diseases are likely to benefit from this procedure?
6. Give examples of any diseases that are already being treated this way, or for which clinical trials will begin soon. Are there any barriers to clinical trials?
7. Suggest a policy for the appropriate use of this gene therapy

Using this organizer, students will participate in a Socratic Seminar to present the information they have learned.

​

More CRISPR resources

Article - 8 Best Videos to Learn More About CRISPR

CRISPR parody video - 7 min

CRISPR animations - recommended by Catherine C.

11/19 -

2.1.1 CQ

2.1.3 - PTC tasting gene - DNA extraction, PCR, and electrophoresis

EQ5 - What is the relationship between phenotype and genotype?

EQ6- What are SNP’s?

​

Read and discuss Intro - we are NOT testing for CF, but doing the same procedure to test for the ability to taste a bitter chemical

Update your Family pedigree

​

Part I - I present next slides, then you complete steps 1-4 in your regular notebook

​

​

Homework -

• In red lab notebook - Lab title, intro, day 1 and 2 materials and procedure which includes DNA extraction and PCR
• 2 articles (1 must be scientific publication about CRISPR) on Gene Therapy Research due Friday

​

Using a Single Nucleotide Polymorphism to Predict Bitter Tasting Ability�Lab Overview

© 2010 Project Lead The Way, Inc.

Medical Interventions

Wed - Step 1: Isolating DNA

• The gene of interest in the experiment, TAS2R38, is located on chromosome #7. This gene is associated with our ability to taste a chemical called PTC.

​

• In the lab, you will isolate a sample of your DNA from your own cheek cells.

​

Thurs: Amplifying the Gene of Interest

• Using your DNA sample, you will amplify a 220 base pair region of the PTC gene using PCR.
• Specific primers attach to either side of the target sequence

​

• In this lab, you will investigate one of the base pair changes or single nucleotide polymorphisms (SNPs) that affects a person’s ability to taste the chemical PTC.

​

​

PCR Review

• show video 1 and 2

Warm-up Question

During PCR, the thermocycler is paused after 5 cycles, 15 cycles, 25 cycles, and 35 cycles. A sample is taken after each of these pauses and the samples are loaded into lanes 2,3,4,5 of the gels seen here. Explain the results.

Genetics Review – Question 1

• The inability to taste PTC is a recessive trait.
• If a capital “T” is is used to designate the dominant allele and a lowercase “t” is used to designate the recessive allele, what is the genotype of a “Nontaster”?

Answer

• A “Nontaster” carries two recessive alleles and thus has the genotype “tt”.

Genetics Review – Question 2

• What are the possible genotypes for a “Taster”?

Answer

• A “Taster” may be homozygous dominant with a genotype of “TT” or heterozygous with a genotype of “Tt”.

​

• In this lab, you will use the tools of molecular biology to determine your genotype for PTC tasting.

​

Monday - Step 3: Restriction Analysis

• Restriction enzymes, molecular scissors, recognize specific DNA sequences and cut the nucleotide strands.

​

​

• In this part of the experiment, you will use a specific restriction enzyme, HaeIII, to identify a SNP or base pair difference in the amplified segment of the PTC tasting gene.

​

Tuesday - Step 4: Gel Electrophoresis

• Gel Electrophoresis separates DNA fragments based on their molecular weight.
• Once you have digested your DNA sample with the restriction enzymes, run your product on a gel to analyze your results.
• Predict the 3 variations...

​

11/20/2018 - 2.1.3 - Extract DNA

• Toothpicks to scrape cheeks and 0.9 % Salt solution or Gatorade
• Always put hinge of tube on outside edge of centrifuge and the pellet will always be directly under hinge
• If pellet size not large enough, can add more spit and re-centrifuge until big enough
• Heat block - time is critical in heat parts
• Keep asking “where is the DNA” and try to follow it - in cells in pellet, but then in supernatant after we’ve lysed our cells

Homework - 2 articles on Gene Therapy Research due Friday, one about CRISPR

​

Me - Take pics of this lab.

11/21 PCR - Amplification of DNA product

​

• PCR tube goes into your pin number in blue ice blocks - LABELED and TIGHTLY CAPPED!!!
• I will run it 45 cycles

​

Once finished, Practice micropipetting and loading gels - buckets with p20’s (1 per person), prac. Gel, Red glycerin water (from Biotech), tips,

• set to 15 ul - check technique - only go to first stop to withdraw and load the gel - do all of yours match?

​

​

Homework - In red lab notebook - Day 3 materials and procedure which includes Digest PCR Product with Restriction Enzyme HaeIII

​

2.1.3 Lab in Red Notebook

1. Intro
2. Day 1 Procedure (DNA Extraction and PCR)
3. Day 2 Procedure (Restriction Digest with HaeIII) - answer questions 11-16
4. Day 3 Procedure (Gel Electrophoresis and Tasting) - answer 24, 30-33
5. Conclusion -
1. Discuss your phenotype and possible genotypes
2. How would you determine each - what would the gel look like?
3. Explain how your genotype determines your phenotype

6) Final flowchart of the lab in your red lab nb, make it clear that you understood why we did all parts and how they interconnected. Where was the DNA and what happened to it for each part?

​

11/22

Last 2 slides, quick check article organizer

Pass in your notebooks open to #1 and your name on it

Guest Speaker

​

​

Homework - Be ready to run Lab day 3 on Monday back

HAVE A GREAT THANKSGIVING BREAK!!!

• Use old tip boxes as racks to hold the tiny PCR tubes
• (Use CyberSafe in gels and buffer) - store in buffer overnight in fridge - Cybersafe stays in dark envelope and put it in a drawer
• TIP - Pull comb out after under buffer in chamber - no bubbles
• Store them in the dark (brown zip lock bags) or just in the fridge
• ​
• ​

​

The following slides are from first two years

Combine DNA extraction and PCR into 1st day. Changed this around with school day cancellation throwing things off a bit in 2018. Also to see if got better PCR results.

11/30 - 2.1.3 - Extract DNA

• Toothpicks to scrape cheeks and 0.9 % Salt solution or Gatorade
• Always put hinge of tube on outside edge of centrifuge and the pellet will always be directly under hinge
• If pellet size not large enough, can add more spit and re-centrifuge until big enough - only once and the
• Heat block - time is critical in heat parts
• Keep asking “where is the DNA” and try to follow it - in cells in pellet, but then in supernatant after we’ve lysed our cells
• Remember PROCEDURE CHANGE - While tube in heat block, DO PCR STEPS 1&2, THEN SPIN (#11) AND PULL 5uL OF CLEAR SUPERNATANT TO ADD TO PCR TUBE
• PCR tube goes into your pin number in blue ice blocks - LABELED and TIGHTLY CAPPED!!!
• I will run it 45 cycles after school

Homework - In red lab notebook - Day 2 materials and procedure which includes Digest PCR Product with Restriction EnzymeHaeIII

• 2 articles on Gene Therapy Research due Friday

​

Me - Must prep lysis buffer today. Take pics of this lab.

• Use old tip boxes as racks to hold the tiny PCR tubes
• (Use CyberSafe in gels and buffer) - store in buffer overnight in fridge - Cybersafe stays in dark envelope and put it in a drawer
• TIP - Pull comb out after under buffer in chamber - no bubbles
• Store them in the dark (brown zip lock bags) or just in the fridge
• ​
• ​

​

12/1 - Restriction digest of PCR product using Hae III

​

2.1.3 - EQ7 - How can restriction enzymes and electrophoresis be used to identify SNPs and determine genotype?

Tips:

• Prep RE right before class - keep frozen/on ice
• Add 1.0 ul of HaeIII - come to me for that - put it directly into the bottom of your PCR tube
• Digest for 2 hours

​

While digesting - set up gel boxes and power supplies

NEED A 2% GEL TO TIGHTEN UP THE BANDS (students get trays ready, I use electric pipetter to fill their trays)

Once gel made, we discuss EQ7 and answer questions 11-16 in your red lab nb

Homework - In red lab notebook - Finish PLTW Part IV questions 11-16, then Day 3 materials and procedure which includes Analyze PCR Product with Gel Electrophoresis and Determine PTC Genotype and Phenotype

2 articles on Gene Therapy Research due Monday

​

​

Me - have flasks of agarose gel solutions made and ready to reheat - 1 per period

2.1.3 Lab in Red Notebook

• Intro
• Day 1 Procedure (DNA Extraction and PCR)
• Day 2 Procedure (Restriction Digest with HaeIII) - answer questions 11-16
• Day 3 Procedure (Gel Electrophoresis and Tasting) - answer 24, 30-33
• Conclusion -
• Discuss your phenotype and possible genotypes
• How would you determine each - what would the gel look like?
• Explain how your genotype determines your phenotype

6) Final flowchart of the lab in your red lab nb, make it clear that you understood why we did all parts and how they interconnected. Where was the DNA and what happened to it for each part?

​

11/27 - from the year they came on Tuesday

1. Introduce Genetic Counseling Grad students from Cal State Stanislaus Genetic Counseling program
2. Assemble case files and review order of presentations
1. 2 printed copies of your written case report
2. 3 printed copies of your patient materials (you keep 1 for your role play and 1 each for myself and guest)
3. 2 rubrics with all of your group member’s names on each

3) 4 minute presentations with 1-2 minute feedback - everyone should be writing in notebook a summary of each case and recommendations

Homework -

2.1.1 will be checked tomorrow - finish CQ

Review PCR with video 1 and 2

2 articles due Friday - 1 of them is scientific about CRISPR