Introduction to PCR

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Back to Micro 5

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### ***Have Students swish outside***

UPDATE THIS FROM THE DOCUMENT THAT IS IN THE PV92 FOLDER IN MICRO 5 FOLDER ON COMPUTER. UPDATED THERE BUT NOT HERE. HARD COPY IN BINDER

### Instructor Notes:

* \*\*\*\*There is enough for 6 groups of 4\*\*\*\*
* On your bench: Clear microtubes of **Master mix / Primer "MMP"** on ice for each bench. The students can come over and scoop some ice and bring their tubes back to their bench after extractions. - I have been warned that if you put it all on their bench, there have been problems with the students mixing up tubes.
* Also on your bench: there are 2 sets of green **control PCR tubes**(++ / +-/ --) - 6 total with 20ul of control templates. Please aliquot 20uL Master Mix / Primers (microtube labeled "MASTER") into each Control PCR tube before putting into the thermal cycler.
* Make sure that students label everything on the tops and sides of every PCR tube They rub off easily and the ink sometimes fades in the heat.
* The students can ignore the letters on the cups.
* There are 3 x 0.9% saline solution bottles. I'll try to get one for each bench next semester!
* I will be in the lab to make sure this is all up to standard and everything runs smoothly, along with bringing the Master Mix and Primers sometime during class.
* Thermalcycler is programmed to PV92

### **Materials**

**Per student**

* 1 x plastic disposable cup
* 1 x 1 mL transfer pipette
* 1 x colored microtube
* 200uL Instagene Matrix in white microtube

**Per bench**

* 10mL graduated cylinder
* 2 x P20 or P200 micropipettors
* 2 x Xcluda micropipette tips
* Beaker of 0.9% Saline solution
* Ice bucket
* Discard Beaker
* Used Tip Beaker

**On side or back bench**

* Vortexer (as many as we have)
* 56 Degree water bath with microtube rack
* boiling 100 Degree water bath with microtube rack
* Microcentrifuge
* Thermalcycler (PROGRAMMED to PV92)

**On Instructor's Bench (For PCR portion of the lab - keep up there so students don't confuse the supplies)**

* Ice bucket
* Microtubes of master mix plus primers (1 for each table)
	+ Add the primers no more than an hour before PCR
	+ Make a tube fore each table - 4 students
	+ Inform the instructor that if they misuse the micropipettors, there may not be enough for all students
	+ See amounts below
* Capless PCR tube holders with PCR tubes
* 3-6 control microtubes on ice ( 20 ul template / 20 ul master mix plus primer)
* tube with extra master mix and primer??

### **Methods**

#### **0.9% Saline Solution**

* Prepare a 0.9% saline solution. To a 500 ml bottle of drinking water, add 4.5 grams of non-iodinated salt. Table salt is recommended. Invert the bottle until the salt goes into solution.

* For each student, place 10 ml saline into a separate cup. Each student workstation should have 4 cups of saline.

#### **Aliquot InstaGene Matrix**

* Thoroughly mix the InstaGene matrix by gently shaking or vortexing the bottle several times to re-suspend the matrix. Be sure that the matrix is well mixed when you aliquot it. The beads settle out of solution quickly, so gently remix the bottle several times during aliquotting.
* Pipette 200 µl of InstaGene matrix into each screw-cap tube. Distribute one tube to each student. Each student workstation should get 4 tubes of matrix for 4 students.
* Have it stirring with stir bar while aliquoting.

#### **Master Mix with Primers**

*Before opening any of the reagent tubes, pulse-spin the contents (~3 seconds) in a centrifuge to bring contents to the bottom of the tubes. Contents often become lodged underneath the caps during shipping.Prepare complete master mix by adding primers. For best results, the following steps should be performed within 15–30 minutes of the PCR reaction.*

* Pipette the master mix into a labeled microcentrifuge tube.

* Label the student microcentrifuge tubes “Master” and place the tubes on ice.
* Add 50 uL MM to 1 uL Primers ratio. See chart below. Vortex 10 seconds to mix. It is imperative that the master mix be evenly mixed after the addition of the primers. The solution should be yellow. The primers are supplied as a concentrated yellow solution in a Tris buffer. Since the primers are much more stable in a concentrated form, add the primers to the master mix just prior to beginning the laboratory exercise — **not more than 30 minutes before the PCR amplification.**
* Aliquot 95 µl of the complete master mix into the student microcentrifuge tubes labeled “Master”, supplying one tube for each student workstation (keep on Instructor Bench on ice). Save the remaining complete master mix for the control reactions. Place these tubes on ice until they will be used.

#### **Control PCR reactions (1 tube for every 4 groups)**

You have to do 1 pcr tube of 40uL's because the Thermal cycler is programmed for that amount. You can't just increase how much, depending on the students.

**Master Mix stuff! - 1 set of 3 controls (++/+-/--) controls for every 4 groups of students**

**You need 20uL of MMP per students and control. Make a bit extra!!!!**

1. Label the control PCR tubes: +/+, –/–, and +/–.
2. Pipet 20 µl of the +/+ template into each +/+ PCR tube.
3. Pipet 20 µl of the –/– template into each –/– PCR tube.
4. Pipet 20 µl of the +/– template into each +/– PCR tube.
5. Pipet 20 µl of the complete master mix into each of the control tubes.
6. Use a fresh tip for each tube.
7. Place the tubes on ice until ready to load into the the thermalcycler.

## Lab 2 Gel Electrophoresis

### **PV92 Special Instructions for Instructor**

1.  Load **10ul** ***Ladder "MWR"** into each **gel***

2.  Aliquot **10ul loading dye "LD"** into each **control**

3. Load **10uL** ***controls*** into each **gel**

4.  Students will aliquot **10uL Loading dye** into each **student sample**

5. Sudents will load **20ul** of ***Student* *Samples*** into **gels**

6.  Run Boxes at 120V for ~30-40 minutes or 2/3 of gel

### **Materials - SPRING 2017 has 7 groups**

#### **At student benches x 7**

* Microtube holder
* 45ul microtube of loading dye (10ul per student)
* P2-20 micropipettes
* Regular micropipette tips

#### **At Instructor's Bench**

* Ice Chest
	+ 6 ++/+-/-- control tubes on ice
	+ Student PCR tubes
	+ Tube of Molecular Marker (original is fine)
	+ Tube of Loading Dye (original is fine)
	+ Ice trays for students
	+ Scoop

**On side bench**

* 7 gel boxes
* 3 power supplies
* 7 x 250 mL TAE buffer (if it isn't in the box yet)
* beakers for aliqotting buffer (2-3)

#### **At Each Sink**

* 1 bottle of Diluted Fast Blast dye
* 1 funnel
* 2-3 dye trays
* Sign telling students to recycle the dye
* Gloves

### **Methods**

#### **Prep TAE Buffer - Sometime before**

* Dilute from 50x buffer to 1x buffer

Each group of 4-5 students will use approximately 320-350 mL of 1x TAE buffer

* for making the agarose gels
* for the gel boxes while running the gels
* So make about 350 \* 7 = 2450mL of 1x TAE

#### **Aliquot Loading Dye**

* Aliquot 45uL of loading dye into a microtube for each group of 4
* Label microtubes "LD"

#### **Making agarose gels - morning of class**

1. Mix TAE buffer and agarose gel to make a 1% running gel
	1. 1 g of agarose to 100mL of 1% TAE buffer
2. Put on hot plate with stir bar until well dissolved (clear-ish)
3. Tape off the ends of the gel trays (1 per group) with lab tape and set on an even surface
4. Put in gel comb (if a group has more than 4 people, use a 10 or 12 tooth comb instead of 8
5. Once the gel is hot and molten, pour into gel tray (it will have a fill line but a little thick isn't bad)
6. Let cool until gel is hardened
7. Let students remove comb and tape and then put into buffer, etc...