Collection of Insects Data

Objective: Students collected insects around Texas to test and see if their insects contained Wolbachia.

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Week 1: March 26-29, 2018

Objective for DNA Extraction:

The students’ objective was to extract DNA from their insect so that they can use the DNA for the polymerase chain reaction to ensure they extracted enough DNA from their insect by using the ALR elution method.

DNA Extraction Protocol:

1. The insects were dissected to remove the gonads.
2. A small sample was taken from the extracted gonads and put in 1.5 mL micro-centrifuge tubes.
3. The tube was labeled by the student’s initials.
4. 75 uL of alkaline lysis reagent was pipetted into the tubes and incubated at 65 degree Celsius for 15 minutes.
5. The tubes were then put in an ice box that was 4 degree Celsius for 5 minutes.
6. After the 5 minutes, 75 uL of neutralizing buffer was added in the tubes.
7. They were then store in the freezer at -20 degrees Celsius.

Objective for PCR:

The students’ objective was to use the DNA extracted earlier and run a polymerase chain reaction (PCR) to ensure their insect obtained DNA by using the COI R and COI F primers.

PCR Protocol:

1. A master mix was made for 25 samples.

Master Mix for Insect DNA

	x1	x25
Nuclease-free Water	12.5 ul	312.5 ul
5X Buffer	5 ul	125 ul
dNTPs	4 uL	100 ul
MgCl2	1.75 uL	43.8 uL
COI F or COI Forward Primer	0.25 uL	6.25 uL
COI R or COI Reverse Primer	0.25 uL	6.25 uL
Taq Polymerase	0.25 uL	6.25 uL

 

2. Each PCR tube was labeled by the student’s initials.
3. 22 uL of the master mix was added into each PCR tube.
4. 3 uL of the student’s insect’s DNA was added to the tube.
5. The tube was vortex for a few seconds.
6. Then the PCR tubes were centrifuged for a few seconds so that the entire solution is at the bottom of the tube.
7. The PCR tubes were then placed into the thermal cycler and put in the “step down” program.

Thermocycler Steps

 

	Step	Duration	Temperature
	Preheat Lid		105 degrees Celsius
1 Cycle of Stage 1	Initial Denaturation	30 minutes	94 degrees Celsius
20 Cycles of Stage 2	Denature	30 seconds	94 degrees Celsius
	Anneal	30 seconds	56 degrees Celsius
	Elongation	1 minute	72 degrees Celsius
20 Cycles of Stage 3	Denature	30 seconds	94 degrees Celsius
	Anneal	30 seconds	56 degrees Celsius
	Elongation	1 minute	72 degrees Celsius
Final Stage	Final Elongation	10 Minutes	72 degrees Celsius
	Hold		10 degrees Celsius

Week 2: April 2-5, 2018

Objective for gel electrophoresis:

Gel Electrophoresis Protocol:

1. Add .3 g of agarose to a 25 mL Erlenmeyer flask

2. Add 30 mL of 1.0x buffer and Swirl it

3. Microwave flask for 30 seconds and swirl to make sure everything is dissolved

4. Microwave flask for 30 seconds again if not clear solution

5. Add1.5 uL of ethidium bromide and swirl flask.

6. Pour the gel into gel tray and then put the comb to make wells.

7.Let the gel sit for 10-15 minutes.

8. Remove the comb and pour .25x buffer into leveling base to the fill line.

9. Use 100 base pair size standard into 1st well.

10. Add ___ uL of gel into PCR product

11. 3 uL of tube inserted to each well.

Objective for PCR:

The students’ objective was to use the DNA extraction from week 1 and run a polymerase chain reaction (PCR) to see if their insect contained the Wolbachia gene by using the Wolbachia R and Wolbachia F primers.

PCR Protocol:

 

1. A master mix was made for 25 samples.

Master Mix for Wolbachia

 

	x1	x25
Nuclease-free Water	12.5 ul	312.5 ul
5X Buffer	5 ul	125 ul
dNTPs	4 uL	100 ul
MgCl2	1.75 uL	43.8 uL
Wolbachia F or Wolbachia Forward Primer	0.25 uL	6.25 uL
Wolbachia R or Wolbachia Reverse Primer	0.25 uL	6.25 uL
Taq Polymerase	0.25 uL	6.25 uL

 

2. Each PCR tube was labeled by the student’s initials.
3. 22 uL of the master mix was added into each PCR tube.
4. 3 uL of the student’s insect’s DNA was added to the tube.
5. The tube was vortex for a few seconds.
6. Then the PCR tubes were centrifuged for a few seconds so that the entire solution is at the bottom of the tube.
7. The PCR tubes were then placed into the thermal cycler and put in the “step down” program.

 

Thermocycler Steps

 

	Step	Duration	Temperature
	Preheat Lid		105 degrees Celsius
1 Cycle of Stage 1	Initial Denaturation	30 minutes	94 degrees Celsius
20 Cycles of Stage 2	Denature	30 seconds	94 degrees Celsius
	Anneal	30 seconds	56 degrees Celsius
	Elongation	1 minute	72 degrees Celsius
20 Cycles of Stage 3	Denature	30 seconds	94 degrees Celsius
	Anneal	30 seconds	56 degrees Celsius
	Elongation	1 minute	72 degrees Celsius
Final Stage	Final Elongation	10 Minutes	72 degrees Celsius
	Hold		10 degrees Celsius

Results

*Insert results here*