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.