Difference between revisions of "GDSYZX-United/NOTEBOOK/protocol"
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<ul role="menu" class="dropdown-menu"> | <ul role="menu" class="dropdown-menu"> | ||
<li><a href="https://2016.igem.org/GDSYZX-United/TEAM/team_members">Team members</a></li> | <li><a href="https://2016.igem.org/GDSYZX-United/TEAM/team_members">Team members</a></li> | ||
| + | <li><a href="https://2016.igem.org/GDSYZX-United/TEAM/collarboration">Collarboration</a></li> | ||
| + | <li><a href="https://2016.igem.org/GDSYZX-United/TEAM/attribution">Attribution</a></li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
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<li><a href="https://2016.igem.org/GDSYZX-United/PROJECT/enzyme_cleavage">Enzyme cleavage</a></li> | <li><a href="https://2016.igem.org/GDSYZX-United/PROJECT/enzyme_cleavage">Enzyme cleavage</a></li> | ||
<li><a href="https://2016.igem.org/GDSYZX-United/PROJECT/preparation_arabidopsis_protoplast">Preparation of the Arabidopsis protoplast</a></li> | <li><a href="https://2016.igem.org/GDSYZX-United/PROJECT/preparation_arabidopsis_protoplast">Preparation of the Arabidopsis protoplast</a></li> | ||
| − | <li><a href="https://2016.igem.org/GDSYZX-United/PROJECT/result">Result</a></li> | + | <li><a href="https://2016.igem.org/GDSYZX-United/PROJECT/result">Result</a></li> |
</ul> | </ul> | ||
</li> | </li> | ||
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<ul role="menu" class="dropdown-menu"> | <ul role="menu" class="dropdown-menu"> | ||
<li><a href="https://2016.igem.org/GDSYZX-United/SAFETY/lab_safety">Lab safety</a></li> | <li><a href="https://2016.igem.org/GDSYZX-United/SAFETY/lab_safety">Lab safety</a></li> | ||
| − | <li><a href="https://2016.igem.org/GDSYZX-United/SAFETY/safety_of_process"> | + | <li><a href="https://2016.igem.org/GDSYZX-United/SAFETY/safety_of_process">Safety of process</a></li> |
</ul> | </ul> | ||
</li> | </li> | ||
Revision as of 13:57, 8 October 2016
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:
-
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 -
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 - 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.
