Protocols for the Methods of DNA
Extraction Used in Chapter Four
DNA EXTRACTION M ETHOD #11 Add 2\i\ of L6 lysis buffer^^i to a sterile 1.5 ml Eppendorf tube containing a single mite.
2 Crush the mite with a pestle made by melting a 1ml pipette tip and moulding it into the bottom of a 1.5ml Eppendorf tube.
3 Mix.
121 L6 lysis buffer
1 Heat 120g o f Guanidinium thiocyanate in 100ml o f O.IM Tris HCl pH6.4 (see below) until dissolved. 2 Add 22ml Of 0.2M EDTA pHS.O (see below).
3 Add 2.6g o f Triton X -100. 4 Mix by vortexing.
5 Store in the dark at room temperature.
0.1 M Tris HCl pH6.4
1 Add 1.21 Ig o f Tris to 100ml o f water. 2 Adjust to pH6.4 with HCl.
3 Store at room temperature.
0.2M EDTA pHS.O
1 Add 8.8ml o f 0.5M EDTA pH8.0 (see below) to 13.2ml o f water. 2 Store at room temperature.
0.5M EDTA pHS.O
1 Add 186.1 g o f Disodium ethylenediaminotetra-acetate.2H20 to 800ml o f water. 2 Stir vigorously with a magnetic stirrer.
3 Adjust the pH to 8.0 with Sodium Hydroxide (about 20g o f NaOH pellets). (The disodium salt o f EDTA w ill not go into solution until the pH o f the solution is adjusted to about 8.0 by the addition o f NaOH (Sambrook, Fritsch and Maniatis, 1989).
4 Dispense into aliquots. 5 Sterilise by autoclaving. 6 Store at room temperature.
4 Incubate at 55°C for 10 minutes. 5 Add 18|il of Sodium Iodide buffer^22
6 Add 2)il of Glassmilk ^ ^3.
7 Incubate at room temperature for 5 minutes vortexing after 2 and 4 minutes. 8 Centrifuge at ISOOOrpm for 5 seconds.
9 Discard supernatant.
10 Add 200|xl of New Wash^^"^. 11 Resuspend.
12 Repeat steps 8-9.
13 Add 400pl of New Wash. 14 Repeat steps 11-13. 15 Repeat steps 11-12.
16 Add 20pl of sterile water to elute. 17 Incubate at 55°C for 5 minutes.
18 Spin down at 13000rpm for at least 30 seconds.
19 Remove supernatant being very careful not to remove any pellet. 20 Add a further 5pl of sterile water to the pellet and resuspend. 21 Repeat step 20.
22 Store extracts at -70°C.
DNA EXTRACTION METHOD #2
As for method 1 but use 1.5pl of L6 lysis buffer at step 1 and 18.5pl of Sodium Iodide buffer at step 5.
DNA EXTRACTION METHOD #3
1 For mites preserved in alcohol, remove the mite from the alcohol and allow to dry thoroughly either in a vacuum drier or covered on the bench at room temperature for about five minutes.
2 Freeze the mite on dry ice for about five minutes or until frozen.
Sodium Iodide buffer
Génecleàn II kit (BIO 101, Cat no. 3106, Stratech Scientific Ltd.). Store in the dark at +4°C.
Glassmilk
Geneclean II kit (BIO 101, Cat no. 3106, Stratech Scientific Ltd.). Store at +4°C.
'24 Wash
Geneclean II kit (BIO 101, Cat no. 3106, Stratech Scientific Ltd.). This must be diluted in ethanol and stored at -20°C and kept on ice whilst in use.
3 Grind up the mite.^^ô
4 When the mite is ground more or less to a powder, remove it from the dry ice and immediately add 40pl of SDS Lysis buffer so that the buffer freezes in the tube. 5 Continue grinding the mite until it is homogenised in the buffer and the buffer has
thawed.
6 Keeping the pestle in the tube add lOpl of 10 mg/ml Proteinase K. 7 Seal the pestle in the tube with parafilm.
8 Incubate at 37°C overnight.
9 Add 150pl 6M Sodium Iodide using it to rinse the outside of the pestle. 10 Discard the pestle.
11 Add 2|il glassmilk.
12 Incubate at room temperature for 5 minutes. 13 Centrifuge at 13000rpm for 5 seconds. 14 Discard the supernatant.
15 Repeat step 13-14 to ensure that all the Nal has been removed. 16 Add 1ml New Wash.
17 Resuspend.
18 Repeat steps 13-14. 19 Repeat steps 16-18. 20 Repeat step 19.
21 Repeat step 13 to ensure that all the New Wash has been removed. 22 Resuspend the glassmilk pellet in 35pl of sterile water.
23 Incubate at 55°C for 5 minutes.
24 Transfer the supernatant to a fresh tube. 25 Add 5|xl of sterile water to the pellet. 26 Repeat step 13.
27 Repeat step 24.
28 Ensure that there is no glassmilk in the extract since very small quantities can inhibit PCR reactions.
29 Store extracts at -70°C.
If the mite thaws during this grinding process place it on dry ice to refreeze.
SDS Lysis buffer
1 M ix 80|il o f water, 100}xl o f O.IM Tris HCl pH6.4 (see above) and 20pl 0.5M EDTA pH8.0 (see above). 2 Adjust the pH to 8.0
3 Add 11.688g NaCl. 4 Add 2g SDS.
5 Store at room temperature.
DNA EXTRACTION METHOD #4
1 Crush frozen mites on dry ice in a 1.5ml Eppendorf tube. 2 Add Ipl of L6 lysis buffer.
3 Incubate at 55°C for 10 minutes. 4 Add 80|xl of Sodium Iodide buffer. 5 Add 2|il of Glassmilk.
6 Incubate at room temperature for 5 minutes vortexing after 2 minutes and after 4 minutes.
7 Centrifuge at 13000rpm for 5 seconds. 8 Discard supernatant.
9 Add 800|il of New Wash.
10 Resuspend the pellet thoroughly. 11 Repeat steps 7-8.
12 Repeat steps 9-11. 13 Repeat step 12.
14 Ensure that as much New Wash as possible has been removed. 15 Resuspend the pellet in 35|xl of water.
16 Incubate at 55°C for 5 minutes.
17 Centrifuge at 13000rpm for 30 seconds. 18 Recover 30|il of supernatant containing DNA. 19 Add a further lOpl of water.
20 Repeat step 18.
21 Recover the remainder of the supernatant. 22 Store extracts at -70°C.
DNA EXTRACTION METHOD #5
As for method 3 but incubate at 55°C for 2 hours at step 8.
DNA EXTRACTION METHOD #6
As for method 3 but incubate at room temperature for 64 hours at step 8.
DNA EXTRACTION METHOD #7