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

Place the tube in a 37℃ incubator, stand for 1 hour.
Water bath the tube in 65℃ for 20 mins to end the digestion reaction.

4.chemical conversion（20160804---20160805）
(1)preparation of LB Broth