Teacher's Guide for Lab DS1:
Precipitating and Spooling DNA
Time of lab
- 35-35 minutes for spooling.
- Lab DS1 is often done in conjunction with other DNA isolations. Look over DS2 (quick and dirty isolation of DNA from calf thymus glands), CF1/2 (reduction in size of DNA by shearing or DNase treatment), and DS3 (long and cleaner isolation of DNA from calf thymus glands) to see if you will want to combine some of these labs.
- DNA can "sit" overnight in buffer to redissolve, then it is ready for the DNase I extension, which will take about 20 minutes without the gel run. If they are running gels, then it will take several days.
- This is an easy activity for an Open House, etc. Plan to set up a "Demo" of the DNase I action.
Preparation before lab
- The solutions can be made several days in advance and refrigerated.
- This activity is a great way to introduce and motivate the student to deoxyribonucleic
acid (DNA). In this lab the DNA has already been isolated from the nuclei of herring
(or salmon) sperm cells.
- Isolated and purified DNA can be obtained from Sigma Chemical Company (1-800-325-3010). The catalog number for salmon sperm DNA is D-1626. Check with Sigma that their current lots of DNAs are of large molecular weight. Some batches of herring sperm DNA have been too low in molecular weight to spool.
- DNA THAT IS SOLD AS A POWDER DOES NOT SPOOL; it must look like matted cotton fibers for good spooling.
- To prepare the DNA solution yourself, place the DNA in sterile TE buffer (10 mM Tris, 1 mM EDTA, pH adjusted to 8.0 with HCI) at a concentration of 3-5 mg/mL for spooling, 10 mg/mL for DNase I demo. It will take 1-5 days to get the DNA into solution. Rock or swirl gently -- do NOT stir or shake. For best longevity, prepare solution in a sterile container with sterile buffer. Store in refrigerator (do not freeze). For a 5 mg/mL solution, dissolve 1 g of DNA in 200 mL TE buffer.
What to Demonstrate/Explain
- Herring fishes belong to the family Cluperidae; a commercially important food fish, Clupea harengus is found in the Atlantic and Pacific waters.
- Remember that, because of the phosphate groups along the "backbone," DNA is negatively charged. Adding the NaCl shields some of these negative charges, allowing the DNA molecules to approach each other and precipitate.
- DNA is not very soluble in 95-100% ethyl alcohol. It is at the interface where the
DNA layer comes in contact with the alcohol layer that the DNA precipitates out of solution and
can be spooled onto a clean glass rod or pipet.