Difference between revisions of "GDSYZX-United/NOTEBOOK/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: (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.