SENO Sperm Sample DNA Extraction Kit

1. Components of the reagent kit

2. Skladištenje

This reagent kit should be stored at room temperature (15-25℃) and in dry conditions, with a shelf life of 12 mjeseca. The DNA extraction purification columns can be stored for 1 year in a cool and dry environment. Proteinase K and RNaza A contain preservatives, allowing transportation at room temperature, but for long-term storage, they should be kept at -20℃.

3. Instructions for Using the Reagent Kit

3.1 This reagent kit is intended for molecular biology research and should not be used for disease diagnosis or treatment.

3.2 Some components in the reagent kit contain irritants. Protective measures such as wearing protective clothing and goggles are recommended.

3.3 During the usage of this reagent kit, a high-speed centrifuge, water bath (metal bath), vortex mixer, anhydrous ethanol, sterile deionized water, and EP tubes need to be prepared by the user.

4. Introduction to the Reagent Kit

The sperm sample genomic DNA extraction kit provides a rapid and efficient purification solution for sperm sample DNA. It achieves sample DNA purification effectively through a dedicated extraction buffer system combined with specific nucleic acid purification columns.

This kit can extract sperm sample DNA within 30 minuta, and the entire purification process does not require toxic reagents such as phenol-chloroform. The extracted DNA can be directly used for PCR, Southern blotting, and other applications.

5. Experimental Principles and Procedures

6. Extraction Process

Before Starting the Experiment:

A.Reagent Buffer SP:This buffer should be stored long-term in an environment between 2℃ and 8℃

B.Reagent Buffer C may precipitate under low-temperature conditions. It is recommended to heat at 65℃ for 5 minuta. After the precipitate dissolves, it can be used normally.

C.Wash Buffer 1: Before use, add the specified amount of anhydrous ethanol as indicated on the reagent bottle label. Mark a check on the label to indicate the addition of anhydrous ethanol.

D. Elution Buffer is a 0.1x TE solution containing a minimal amount of EDTA. If EDTA has an impact on subsequent experiments, it is recommended to use sterile deionized water as a substitute for the elution buffer.

1. Sample Processing:

Pipeta 200μl of frozen sperm, incubate at 75℃ for 10 minutes until the sperm sample is not viscous. Centrifuge at 12000 rpm for 5 minuta, aspirate the supernatant as much as possible, and the sperm cells will sediment at the bottom of the tube.

2. Add to the sediment from the previous step: 400μl Reagent Buffer SP, 20μl Proteinase K (10 mg/ml), 20μl RNaseA (10 mg/ml).Digest at 65℃ for 10 minuta, invert and mix 6-7 times during digestion until the sample is fully digested.
3. Add an equal volume of Reagent Buffer C and an equal volume of anhydrous ethanol, and mix well by pipetting.

(For example: If you add 400μl of Reagent Buffer IV, you will need to add approximately 460μl of Reagent Buffer C and 460μl of anhydrous ethanol. A small amount of precipitation may form after adding Reagent Buffer C, but it does not affect subsequent experiments.)

4. Transfer the obtained liquid to a DNA extraction purification column (cassette) (approximately 650-700μl each time). Let it stand at room temperature for 2 minuta, centrifuge at over 8,000 rpm for 1 minute, discard the collected waste liquid, and re-insert the collection tube into the purification column for the next step.
5. Repeat step 4, adding the remaining liquid to the DNA extraction purification column (cassette), centrifuge at over 8,000 rpm for 1 minute, discard the waste liquid and the collection tube.
6. Place the DNA extraction purification column (cassette) into the collection tube, add 300 μl of Wash Buffer 1, centrifuge at over 8,000 rpm for 1 minute, discard the waste liquid, and place the DNA extraction purification column (cassette) back into the tube for the next step.

(Note: Confirm that anhydrous ethanol has been added to Wash Buffer 1.)

7. Dodati 500μl of Wash Buffer 1 to the DNA extraction purification column (cassette), centrifuge at 14,000 rpm (20,000×g) za 2 minuta, extend the centrifugation time appropriately to dry the membrane more thoroughly.
8. Place the DNA extraction purification column (cassette) into a new centrifuge tube, open the lid, incubate at 65℃for 2 minuta. Extend this step if necessary to evaporate ethanol as much as possible, preventing ethanol residue from affecting downstream experiments.
9. Drop-suspend 50-100μl of Elution Buffer onto the membrane, centrifuge at 12,000 rpm for 2 minuta.

(Note: 1. Using 50μl of Elution Buffer to elute DNA can increase DNA concentration but decreases the overall DNA yield. 2. The eluted DNA can be reapplied to the DNA extraction purification column, centrifuged at 12,000 rpm for 2 minutes again, to increase DNA yield.)