"
Team:UESTC-China/Protocol
From 2014.igem.org
(Difference between revisions)
Zhangmanni (Talk | contribs) |
Zhangmanni (Talk | contribs) |
||
| Line 337: | Line 337: | ||
line-height:40px; | line-height:40px; | ||
font-size:24px; font-family: calibri, arial, helvetica, sans-serif; border-bottom:0px; color:#ffffff; font-weight: bold;font-style: Italic; text-align:center; width:1140px; } | font-size:24px; font-family: calibri, arial, helvetica, sans-serif; border-bottom:0px; color:#ffffff; font-weight: bold;font-style: Italic; text-align:center; width:1140px; } | ||
| + | |||
#SectionTitles2{ | #SectionTitles2{ | ||
position:relative; | position:relative; | ||
| Line 526: | Line 527: | ||
</table> | </table> | ||
</div> | </div> | ||
| - | |||
<div id="SectionTitles2" style="width:500px;">DNA ligation with Gibson Assemble | <div id="SectionTitles2" style="width:500px;">DNA ligation with Gibson Assemble | ||
</h1> | </h1> | ||
| Line 704: | Line 704: | ||
</table> | </table> | ||
| - | </div> | + | </div> </div> |
| + | |||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 05:53, 24 September 2014
DNA ligation with T4 DNA Ligase
| Component | 20ul Reaction |
|---|---|
| 1.33X Master Mix | 15ul |
| Insert DNA | the moles of insert DNA to vector DNA is 5：1 |
| >Vector DNA | 50ng |
| ddH2O | To 20ul |
| Incubate at 50℃ for 1 hour |
DNA ligation with Gibson Assemble
| Component | 20ul Reaction |
|---|---|
| Vector DNA | 50ng |
| Insert DNA | the moles of insert DNA to vector DNA is 5：1 |
| 10X T4 DNA ligase buffer | 2ul |
| T4 DNA ligase | 1ul |
| ddH2O | to 20ul |
| Incubate at 25℃ for 12 hours |
Double Digestion of DNA
| Component | 50ul Reaction |
|---|---|
| 10x FastDigest buffer | 5ul |
| Restriction enzyme 1 | 1ul |
| Restriction enzyme 2 | 1ul |
| DNA | 1-2ug |
| ddH2O | to 50ul |
| Incubate at 37℃ for 2 hours |
PCR protocol for KOD-Plus-Neo
| Component | 50ul Reaction |
|---|---|
| ddH2O | 33ul |
| 10X buffer for KOD-Plus-Neo | 5ul |
| MgSO4 | 1ul |
| 10mM dNTPs | 1ug |
| 10uM forward primer | 1ul |
| 10uM reverse primer | 1ul |
| Template | 1ul |
| KOD enzyme | 1ul |
| Temperature | Time | Cycle | |
|---|---|---|---|
| Step1 | 94℃ | 30s | X35 cycles |
| 56℃ | 30s | ||
| 68℃ | 30s/kb | ||
| Step3 | 68℃ | 5min | X1 cycles |
| 10℃ | 10min | ||
| 10uM reverse primer | 1ul | ddH2O | 33ul |
