|
|
Line 4: |
Line 4: |
| <center>'''-----EcoRI--XbaI--Part--SpeI--PstI-----'''</center> | | <center>'''-----EcoRI--XbaI--Part--SpeI--PstI-----'''</center> |
|
| |
|
| This is what Mike calls "Standard Assembly 2.0" and is a method of "bio-bricking" two parts together. For more information on bio-bricking see this link. This method combines the ease of standard assembly with the fidelity of triple-antibiotic (3A) assembly. The basic steps are as follows. Your two parts will be thusly labeled "insert" and "vector" although initially they will both be contained on separate plasmids.
| | See the biobrick assembly schedule for more information on using this technique. |
| | |
| #Miniprep both "insert" and "vector" from their respective cultures.
| |
| #PCR the insert plasmid using a high-fidelity polymerase and the same primers you use for colony PCR.
| |
| #Purify the PCR product.
| |
| #Digest the both vector and PCR'd insert according to the instructions in this protocol.
| |
| #Add DpnI restriction endonuclease to the "insert" digest.
| |
| #Add Antarctic Phosphatase to the "vector" digest.
| |
| #Ligate
| |
| #Transform
| |
| #Celebrate
| |
|
| |
|
| ==Materials== | | ==Materials== |