Difference between revisions of "Team:UGent Belgium/SOP"
m |
|||
| Line 10: | Line 10: | ||
<html> | <html> | ||
| − | |||
<div class="container"> | <div class="container"> | ||
| − | <h2> | + | <h2>Protocols used</h2> |
| + | |||
<p>Under construction.</p> | <p>Under construction.</p> | ||
| Line 69: | Line 69: | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
| − | <p>The tubes are incubated for 30 sec. at 98℃, 15 cycles of (10 sec. at ℃; 30 sec. at 55℃; construct length(kb) x 15 sec. at 72℃) and 10 min. at 72 ℃. | + | <p>The tubes are incubated for 30 sec. at 98℃, 15 cycles of (10 sec. at 98℃; 30 sec. at 55℃; construct length(kb) x 15 sec. at 72℃) and 10 min. at 72 ℃. |
1-2uL is then used directly for transformation, being careful not to add more as this increases the risk of arcing.</p> | 1-2uL is then used directly for transformation, being careful not to add more as this increases the risk of arcing.</p> | ||
| Line 127: | Line 127: | ||
</ol></p> | </ol></p> | ||
</div> | </div> | ||
| − | |||
Revision as of 21:34, 17 October 2016
Protocols used
Under construction.
PCR
Polymerase Chain Reation (PCR) was used extensively during both the assembly and verification of new constructs. For assembly work, we used high-fidelity DNA polymerases: PrimeSTAR HS, PrimeSTAR GXL (TaKaRa Bio) and Q5 (New England Biolabs). The manufacturer's instructions were followed unless mentioned otherwise. To create linear fragments suitable for assembly, BsaI restriction sites or overlapping sequences were added to the 5' end of the primers during the in silico design. The length of each fragment was verified using agarose gel electrophoresis, reaction mixture were purified using a PCR purification kit (Analytik Jena) and DNA concentration determined using a NanoDrop device (Thermo Scientific).
Verification of construct assembly was mostly performed using Colony PCR with Taq DNA Polymerase or OneTaq Quick-Load Master Mix. Colonies were randomly selected and picked by hand using sterile pipette tips, each which was then briefly touched to a back-up plate and placed in a PCR tube. We slightly modified the manufacturer's protocol by extending the initial denaturation time to 2 minutes, to ensure lysis of the colonies. The length of each fragment was again determined by agarose gel electrophoresis.
Circular Polymerase Extension Cloning (CPEC)
Shorter constructs such as the plasmids carrying our basic parts, as well as the empty cloning vectors, were assembled from linear backbones created by PCR and synthetic fragments (gBlocks) using Circular Polymerase Extension Cloning (CPEC)[1]. Our version of the protocol makes use of Q5 polymerase in the following reaction mixture:
| Component | Amount |
|---|---|
| Vector backbone | 100-200 ng |
| Insert | 3:1 molar ratio to the vector |
| Q5 buffer | 5.0 uL |
| dNTPs (2mM) | 5.0 uL |
| DMSO | 0.75 uL |
| Q5 DNA polymerase | 0.5 uL |
| dH2O (add first) | to 15uL total volume |
The tubes are incubated for 30 sec. at 98℃, 15 cycles of (10 sec. at 98℃; 30 sec. at 55℃; construct length(kb) x 15 sec. at 72℃) and 10 min. at 72 ℃. 1-2uL is then used directly for transformation, being careful not to add more as this increases the risk of arcing.
Golden Gate Cloning
Golden Gate cloning [2] was used to efficiently and seamlessly assemble and clone linear fragments into our expression vectors (BBa_K1896019 and BBa_K1896020). 15uL reactions are set up using NEB enzymes according to the table below. The tubes are then incubated for 25 cycles of (3 min. at 37℃; 4 min. at 16℃), 10 min. at 50℃ and 10 min. at 80℃, after which 2uL is used for transformation, without purification.
| Component | Amount |
|---|---|
| Vector DNA | 100 ng |
| Linear pieces | 3:1 molar ratio to the vector |
| T4 buffer | 1.5 uL |
| BSA (100x) | 2.0 uL |
| BsaI-HF | 1.0 uL |
| T4 DNA ligase | 1.0 uL (400 units) |
| dH2O (add first) | to 15uL total volume |
Transformation
Plasmid isolation
Protein extraction
Native PAGE
References
- Quan, J., & Tian, J. (2009). Circular polymerase extension cloning of complex gene libraries and pathways. PloS one, 4(7), e6441.
- Engler, C., Gruetzner, R., Kandzia, R., & Marillonnet, S. (2009). Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PloS one, 4(5), e5553.
