GDSYZX-United/NOTEBOOK/protocol

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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: (view primer sequence in PDF)

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.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.