I.           Extraction of Genomic DNA

A.         WLB (Worm lysis buffer): after Williams et al., 1994 Genetics 131:60

 

WLB (Worm lysis buffer):

No.	Chemicals	F.W.	Stock	Final	1  ml	2  ml	5  ml
1	KCl (Sigma, P-9541)	74.56g	1M	50 mM	50 µl	100 µl	250 µl
2	Gelatin* (Dicto Bacto, 0143-02 ¼ lb)		1%	0.05 %	50 µl	100 µl	250 µl
3	Tris pH 8.2 (BioRad, 161-0719)	121.14g	1M	10 mM	10 µl	20 µl	50 µl
4	Tween 20 (Fisher, FL-04-0796)	1227.54g	100%	0.45 %	4.5 µl	9 µl	22.5 µl
5	Proteinase K (Roche, 0 092 766, 1gm)		20mg/ml	60µg/ml	3.3 µl	6.6 µl	16.5 µl
6	MgCl2 (From PCR reagents)	95.21g	1M	2.5 mM	2.5 µl	5 µl	12.5 µl
7	ddH2O				880 µl	1760 µl	4400 µl

(*: made fresh, 100mg gelatin in 10ml water and heat in microwave)

1.         Many worms                                                

1. Add 50 µl lysis buffer to tissue sample (approximately 0.5 cm) in a 0.5 ml PCR tube.
2. Place at -70ºC > 15min. Can store for several days. Or put in liquid nitrogen to 55ºC water bath for 10 times to help break down the nematode body.
3. Warm to room temperature and add 1 drop mineral oil.
4. Incubate at 60ºC > 1 hour. Vortex at least once during incubation to help breakup tissue.
5. Heat to 95ºC for 15 min. (Kills off Proteinase K).
6. Cool to 4ºC.
7. Vortex briefly (2-3 sec).
8. Spin at 6,000 rpm for 30 sec.
9. Use 1 µl supernatant as template for PCR amplification for 25 µl reaction. 

2.     Nematodes-for single worm

1. Add 15 µl lysis buffer to worm in a 0.5 ml PCR tube.
2. Place at -70ºC > 15 minutes. Can store for several days.
3. Warm sample to room temperature and add mineral oil.
4. Incubate at 60ºC > 1 hour.

e.     Heat to 95ºC for 15 minutes

f.      Cool to 4ºC.

g.     Pipet sample up and down to mix

h.     Use 2.5 µl as template for PCR amplification.

 

B.        DNA extraction using Geneclean III

 

·       Turn on centrifuge to 4ºC, turn on waterbath to 58ºC, turn on rice cooker, take out Proteinase K from freezer to thaw.

1.     Lysis

1. Transfer droplet containing nematodes stored in 1M NaCl to clean slide (Cleaned with 70% ethanol and wiped with kimwipe).
2. Transfer 1 nematode by a needle picker to 1.5ml microtube containing 18 µl Lysis Buffer.
3. Crush nematode using pipette tip for about 40 times.
4. Spin at 13,000 rpm for 1 min to bring solution down to bottom of the tube.
5. Freeze at -20ºC for 20 min.
6. Boil for 5 min in rice cooker, after 1 min in boiling water bath, open tube caps and reclose to release pressure build-up.

2.     Proteinase K digestion

a.     Burst spin.

b.     Add 2µl Proteinase K (20 mg/ml stock) and vortex.

c.     Incubate at 58ºC for 3 hours.

d.     Turn on waterbath down to 51ºC

3.     Gene clean

1. Burst spin.
2. Add 60µl NaI solution and vortex.
3. Add 0.8µl EZ-Glassmilk and vortex.

Incubate at room temperature for 5 min.

Spin for 30 seconds at full speed and remove supernatant.

4. Add 60µl New Wash and vortex to resuspend all EZ-Glassmilk.

Burst spin and remove supernatant.

Repeat New Wash 2 more times.

Spin for 30 seconds at full speed and remove supernatant.

5. Leave the cap open for 10 min at room temperature or place the tube under vacuum for 2-5 min.
6. Add 10µl GCIII Elution and resuspend EZ-Glassmilk.

Incubate at 51ºC for 3 min.

Spin for 30 seconds at full speed to make a solid pellet and collect supernatant into a new 0.5ml microtube.

7. Add 5µl GCIII Elution and resuspend EZ-Glassmilk.

Incubate at 51ºC for 3 min.

Spin for 30 seconds at full speed to make a solid pellet and collect supernatant into the previous microtube.

8. Spin for 30 seconds at full speed again to get rid of the glass milk and collect supernatant into a new microtube
9. Straight to PCR or freeze at -80ºC until ready.

C.        Non-manual lysis after Stanton et al., 1998 Australasian Plant Pathology 27:112.

 

1. Pick the single into microtube containing 10µl 0.25M NaOH, crash with pestle, then add 10µl 0.25M NaOH.
2. Incubate at 25ºC for 24 hours.
3. Further incubate in a thermocycler at 99ºC for 2 min.
4. 10µl 0.25M HCl, 5µl 0.5M Tris-HCl, pH8.0, and 5µl 2% Triton X-100 were added and incubated for another 2 min.
5. Straight to PCR or freeze at -80ºC until ready.

 

 

Example SAMPLE TRACKING SHEET (simple format)

#	Species	PCR	Remark		#	Species	PCR	Remark
1					11
2					12
3					13
4					14
5					15
6					16
7					17
8					18