User:Abass Conteh/Notebook/Analysis of CRIPT mutants/Entry Base

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Screening Salk T-DNA Mutants

Purpose: Identify plants with T-DNA insert and determine their genotypes.

Extracting Plant DNA

Materials and Reagents Needed:

  DNA extraction materials:
   1. Extraction buffer: 
       200 mM Tris-HCl (pH 8.0)
       200 mM NaCl
       25 mM EDTA
       0.5% SDS.
  2. Sample leaf

 12 µM Gene-specific Forward primer 
 12 µM Gene-specific Reverse primer 
 12 µM LBb1 primer 
 10X Ex-Taq buffer 
 dNTP Mix 
 Ex-Taq DNA polymerase 
 Sterile water 
 1-kb DNA ladder 
 PCR Machine  
 0.2 mL PCR tubes 
 1.5 mL microcentrifuge tubes 
 P-10, P-20, P-200 Pipet 
 PCR rack for 0.2 mL PCR tubes 
 Rack for 1.5 mL microcentrifuge tubes 
 Filtered Pipet tips for PCR 
 Ice bucket 
 Gel apparatus and power supply 
 Gel Documentation System