PART I. Genomic DNA isolation from sea urchin sperm
I-1. The Inexpensive way (from Zeev Pancer)
1. Resuspend 25-50ul (or 100 – 200 ul) of fresh sea urchin sperm in 1ml 1XSSC, spin down at 3-5k
2. Resuspend a pellet of 100-200ul sperm in 15ml TE; add 0.75ml of 20%SDS, 0.5m of b-mercaptoethanol, and 0.5ml of proteinase K (20mg/ml stock).
3. Mix by inversion and incubate at 56C for 2hr; mix by inversion several times
4. Extract twice with equal volumes of PCIA (Phenol/Chloroform/Isoamulalcohol); rotate for 0.5hr on a wheel), then extract once with CIA.
5. Add 0.1 volume of 3M NaAc(pH 5-6) and 2 volumes EtOH; mix and spool out the DNA on sealed glass capillaries
6. Wash briefly by immersion into 5ml 70% EtOH, air dry nriefly and resuspend in 1-5ml of 1-0.5xTE. May require time to dissolve completely.
I-2. The Expensive way (from Sarar Damle)
1) start with 25-50ul sperm cells
2) wash w/ 1ml 1xssc, spin 4krpm, 5′
3) repeat once
4) use DNA isolation kit from Qiagen (Blood & Cell culture DNA MAxi kit). Cat# 13362 (protocol for cell cultures). Perform the lysis step on p.25
5) use the same kit. Perform the DNA purification step (p.44)
* It may be better to use two columns for every 25ul of sperm used. I feel that I was overloading when I used just 1 column.
* I did not spool DNA after elution. I just centrifuged (see steps 5B, 6B)
PART II. DNA digestion
Genomic DNA digestion to make carrier
1) digest 10ug genomic DNA using HindIII
*200 units HindIII
*100 ul reaction
* digest 8hrs @ 37C
2) add a second aliquot of enzymes and buffer and digest O/N @ 37C (200ul final volume)
3) proteinase K treatment (50ug/ml final concentration), 30′, RT
4) phenol chloroform extraction
5) O/N Ethanol precipitation (isopropanol precipitation works too)