Difference between revisions of "GDSYZX-United/NOTEBOOK/protocol"
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Revision as of 05:14, 26 September 2016
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.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.
