GDSYZX-United/NOTEBOOK/protocol

Protocol

1.Extract Arabidopsis genomic DNA
Material:
  • EZ-10 Spin Column Plant Genomic DNA Purification Kit
  • β-mercaptoethanol
  • RNaseA
  • Chloroform
  • Ice box and ice
2.PCR
Material:
  • ddH20
  • dNTPs mix
  • 10×Buffer
  • Taq polymerase
  • PCR Primers
  • Arabidopsis genomic DNA
Methods:
  1. add material into a 0.2ml EP tube according to the table
    Material dosage/μl
    dNTPs mix 1
    Forward primer 0.5
    Reverse primer 0.5
    Arabidopsis genomic DNA 3
    ddH20 12
    Taq polymerase 0.5
    Total 20
  2. set the PCR program
    • For promoter cop1,phyB,pif1,gene hhl1,gene flu:
      94℃ × 5min +(94℃×30s +Tm×30s +72℃×1min)×35+72℃×5min +4℃(for preserving)
    • For pHhl1-gene hhl1:
      94℃ × 5min +(94℃×30s +Tm ×30s +72℃×90s) ×30+72℃×5min +4℃(for preserving)
    • Tm of each primer:
      Name Tm(Forward/Reverse)(℃) Tm in PCR(℃)
      Promoter pif1 62.59/62.67 63
      Promoter cop1 66.77/65.46 66
      Promoter phyB 63.94/63.90 64
      pHhl1-genehhl1 60.75/59.38 60
      gene hhl1 60.90/59.38 60
      Fluorescent gene flu 62.42/66.90 65
3.digestion
Material:
  • ddH20
  • BsaⅠBuffer
  • PstⅠBuffer
  • restriction enzyme BsaⅠ，PstⅠ，EcorⅠ
  • PstⅠBuffer:100mM NaCl、50mMTris-Hcl、10mM MgCl2、100μg/mlBSA、ph7.9
  • Bsa1 Buffer:50mM CH3COONa、20mMTris-Hcl、10mM(CH3COO)2Mg、100μg/mlBSA、ph7.9
Method
  1. 1.add materials into a 1.5ml EP tube according to the table below:
    Material dosage/μl
    PCR product(after purification) 17
    restriction enzyme buffer 3 (/4)
    restriction enzyme 1(0.5 each)
    ddH20 4
    Total 25

    PCR product cutting sites restriction enzyme Buffer
    pHhl1-hhl1 BsaⅠ+EcorⅠ 3μlPstⅠ+1μlBsaⅠ
    hhl1 PstⅠ+EcorⅠ 3μlPstⅠ
    flu EcorⅠ+BsaⅠ 3μlPstⅠ+1μlBsa1Ⅰ
    cop1 BsaⅠ+PstⅠ 3μlPstⅠ
    phyB BsaⅠ+PstⅠ 3μlPstⅠ
    pif1 BsaⅠ+PstⅠ 3μlPstⅠ
  2. Place the tube in a 37℃ incubator, stand for 1 hour.
  3. Water bath the tube in 65℃ for 20 mins to end the digestion reaction.