GMO Lab

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PCR LAB NO LONGER DONE

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## Lab 1 Extraction and PCR

### *Can prepare beforehand and store in fridge*

1. White Microtubes = 2 / bench *(Can prepare and aliquot beforehand and store in fridge for as long as needed)*
	* 500ul Instagene Matrix in each white microtube
	* Label 1 =  “**NON-GMO**” | Label 2 = “**Test**”
	* Make sure to keep it stirring with a mini stir bar as you aliquot to microtubes
	* We will use regular pop-caps but tell the instructors to watch them in the bath so the tops don't pop off.

1. Yellow Microtubes "+ Control" *(Can do ahead of time and freeze)*
	* 1 yellow microtube / bench
	* Label yellow microtubes "+ Control"
	* Aliquot 45uL of + control from Bio Rad tube into each microtube
	* Put on ice

### *To make in a batch no more than 1 hour before class*

1. GREEN microtube “PMM” Plant Master Mix WITH PRIMERS
	* Make master batch for class: (# of groups)
		+ 65uL x # of groups + 25 extra = ? uL master mix with primer
	* 50:1 master mix to primer ratio = total ? ul PMM / 50 = **? uL primers + ? uL master mix**
	* Aliquot 65 uL into green microtubes, label "**PMM**" = 1/bench

1. PINK microtube “GMM” GMO Master Mix WITH PRIMERS
	* Make master batch for class: (# of groups)
		+ 65uL x # of groups + 25 extra = ? uL master mix with primer
	* 50:1 master mix to primer ratio = total ? ul GMM / 50 = **? uL primers + ? uL master mix**
	* Aliquot 65 uL into green microtubes, label "**GMM**" = 1/bench
	* Put on Ice

### *Setup Classroom*

#### **Front Bench**

* Ice chest containing the reagents below
* white microtubes of Instagene Matrix = 1/bench

* Master Mix and Primer tubes
	+ Green microtubes: 65uL master mix and plant primer labeled "**PMM**" = 1/bench
	+ Pink microtubes: 65uL master mix and Bt GMO primer labeled "**GMM**" = 1/bench
	+ Yellow microtubes: 43uL positive control labeled "**+ Control**" = 1/bench

* A micropipette and box of tips with instructions on how to use a micropipette
* Microtube rack
	+ 6 PCR tubes (3 red and 3 green) = 1 set per bench of 6 tubes

#### **Student Benches**

* 2 x 500ul Instagene Matrix in screw cap TTs - \*Label 1 =  “**NON-GMO**” / Label 2 = “**Test**”
	+ Make sure to keep it stirring with a mini stir bar as you aliquot to microtubes
	+ We will use regular pop-caps but tell the instructors to watch them in the bath so the tops don't pop off.
	+ Can prepare and aliquot beforehand and store in fridge

* P200 Micropipette and Tips
* ~25mL + beaker of distilled water
* Pestle and mortar
* Sharpie
* Microtube rack
* P200 micropipettor
* PCR tips (1 box - maybe put on instructor bench)
* Regular Tips (for practice)
* Tip disposal beaker
* Instructions on how to use a micropipette

### **Side and back benches**

* Balances = 1/bench
* Weigh boats 2/bench
* at least 2 g certified non GMO food control sample per bench (oatmeal in kit)
* at least 2 g test food sample per bench (they bring their own) = no salt corn chips, can have claim of no GMO can not have claim - one of each at least
* 95 degree Celsius water bath with microtube holders (for 24 tubes)
* Centrifuge
* Thermal cycler programmed for GMO lab
* Dish soap and sponge at each sink

## Lab 2: Gel Electrophoresis and Analysis

**All of these recipes will be for EACH CLASS = 6 groups**

### *Make and dilute TAE buffer:*

1. Make stock 1x TAE buffer (look up on internet for recipe)
	* Store 1x TAE in buffer cupboard as stock solution
	* Before use, dilute 45mL of 50x TAE buffer with 2205mL DI water in large jug

### *Agarose gels*

* It is recommended to do a **3% concentration** for agarose on this, to more quickly separate the DNA.
* 3g of agarose to 100mL 1 x TAE Buffer
* heat and stir until molten and all that
* each gel box will get about 45mL agarose gel = 45mL x # of groups
* Can store in plastic bags in refrigerator for 1-2 days

### *Fast Blast Gel Stain*

* Dilute 500x to 100x Fast Blast stain
* Can be reused for about 7 semesters
* Keep in media bottles labeled with use dates
* Instruct students to return the dye into bottles after use

### *Add Loading Dye to student Samples*

* Add 10uL of orange loading dye to all student PCR samples
* Add 40 uL to 1 vial of molecular weight vial ( for every 200 uL MWL = 240 uL Total)
	+ Each group needs 20 uL ( x 6 = 120 uL total volume)
	+ May not need to add loading dye if the MWR is already colored

### *Setup the classroom*

#### **On Student Benches**

* Ice dish
	+ The student's PCR products with pre-added loading dye
	+ 25ul molecular weight ruler labeled "MWR" (Can also just have the original MWR tube on back bench for students to share)
* laminated piece of white paper (for viewing gel)

#### **On back bench**

* Casted agarose gels
* Gel boxes with tops
* Power packs
* Jug of 1x TAE buffer
* Beakers for students to dispense TAE buffer
* sandwich bags for storage of gels
* P20 micropipettors
* P20 Tips
* Disposal Beakers

#### **At each sink**

* Dye trays
* 1x media bottle of 100x fast blast DNA stain
* Buckets in sink labeled with rinse times
* timers
* Fast Blast Dye dispense container and funnel
* Sign to tell students to return dye into bottle