Restriction Digest: Partial

Abstract

This protocol can be used to generate genomic DNA fragments of different sizes using a common cutter. This is useful for many screening/probing processes.

Materials

• 10 microcentrifuge tubes

Reagents

• Restriction Endonuclease
• 10X Restriction Endonuclease buffer
• BSA (10 mg/mL)
• Genomic DNA (Concentration > 0.1 μg/μL)

Procedure

1. Label the microcentrifuge tubes 1 - 10
2. Mix together the following and invert to mix:
   1. 180μL Genomic DNA
   2. 20μL 10X Buffer
   3. 2μL BSA
3. Dispense 40μL of this mix into tube 1 and 20μL into tubes 2 - 10 **From this point keep everything on ice!!!
4. Add 1μL of restriction-endonuclease to tube 1 and invert to mix.
5. Transfer 20μL from tube 1 to tube 2 and invert to mix.
6. Repeat the last step transfering 20μL from the preceding tube to the succeeding tube mixing with every transfer.
7. Finally remove 20μL from tube 10
8. Incubate all 10 tubes for 1 hour at 37°C.
9. Finish the digest by submerging all 10 tubes in an 70°C water bath for 20 minutes.
10. Run the samples on a gel and choose the size which best fits your application.

Acknowledgments

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