III. Purification of PCR products.

A.Solid Phase Reversible Immobilization (SPRI) Purification of PCR Products

1. Sample Sheet Plate name:

Gene
	1	2	3	4	5	6	7	8	9	10	11	12
A	1	9	17	25	33	41	49	57	65	73	81	89
B	2	10	18	26	34	42	50	58	66	74	82	90
C	3	11	19	27	35	43	51	59	67	75	83	91
D	4	12	20	28	36	44	52	60	68	76	84	92
E	5	13	21	29	37	45	53	61	69	77	85	93
F	6	14	22	30	38	46	54	62	70	78	86	94
G	7	15	23	31	39	47	55	63	71	79	87	95
H	8	16	24	32	40	48	56	64	72	80	88	96

2. Preparation of magnetic beads

1. Dilute beads (Eastapor SuperParMagnetic Microspheres [ME03N, Bangs Laboratories, INC, 317-5707020, Fax 317-5707034]) from bottle at 1:5 in 0.5M EDTA (pH8.0): 2 tubes 10ml each using 2ml beads, 8ml EDTA.
2. Wash beads 3 times with 0.5M EDTA, using magnetic separation plate.
3. Thoroughly resuspend beads prior to use.

3. Protocol

1. Transfer PCR samples to plate (Falcon 353911).
2. Add 50µl hybridization buffer (2.5M NaCl, 20% PEG8000) and 10µl washed beads to each 50µl PCR reaction, resuspend mixture well.
3. Incubate 10 minutes at room temperature. Resuspend mixture well again.
4. Place tubes on magnetic separation plate, allow to separate for 3 minutes or until beads have completely separated.
5. Remove supernatant from tubes using aspirator and discard.
6. Wash bead pellet with 150µl 70% EtOH while still on magnetic plate, repeat wash once.
7. Allow beads to air dry for 1 hour. (starting time:_________, finish time:________). (it helps to invert plates)
8. Add 50µl elution buffer (10mM Tris) to each well, thoroughly resuspend bead pellet. Save tips.
9. Incubate 3 minutes at room temperature. Resuspend mixture wel, place tubes on magnetic separator.

1. Allow beads to separate 5 minutes, remove supernatant (contains PCR product) to a new plate. Store at –20 degrees C.
2. Prepare 4µl each sample to a plate for gel electrophoresis.

B. QIAquick Gel Extraction Kit Protocol

• Cat. No. 28704 ($61.20 for 50 samples), Qiagen, Made in Germany
• Turn the water bath to 50ºC, make 1% agarose gel.

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Our 1.5ml tube is 1.1 g/each. Add 3 volumes of Buffer QG to 1 volume of gel (Usually 500ul).
3. Incubate at 50ºC for 10 min. To help dissolve gel, mix by vortexing the tube every 2-3 min during the incubation.
4. Add 1 gel volume of isopropanol to the sample and mix (If DNA<500bp or >4kb).
5. Place a QIAquick spin column in a provided 2ml collection tube.
6. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
7. Discard flow-through and place QIAquick column back in the same collection tube.
8. Add 0.5ml of Buffer QG to QIAquick column and centrifuge for 1 min.
9. To wash, add 0.75ml of Buffer PE to QIAquick column, let the column stand 3 min and centrifuge for 1 min.
10. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 13,000rpm.
11. Place QIAquick column into a clean 1.5ml microfuge tube.
12. To elute DNA, add 20µl water to the center of the QIAquick membrane, let the column stand 3 min and centrifuge the column for 1 min at 13,000rpm.
13. Store the sample at -20ºC freezer.

Example sample Sheet:

No.	PCR#	Sample#	Species	bp	ng/µl	Result
1
2
3
4
5
6
7
8