Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics/Protocols/MaSp ICA Fragments"
Vinsonclam (Talk | contribs) (→BsaI Digestion) |
Vinsonclam (Talk | contribs) (→Maxiprep (Cloning Amplification)) |
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===Maxiprep (Cloning Amplification)=== | ===Maxiprep (Cloning Amplification)=== | ||
*Streak the glycerol stocks on chloramphenicol plates, and grow overnight at 37 C. | *Streak the glycerol stocks on chloramphenicol plates, and grow overnight at 37 C. | ||
− | *Pick | + | *Pick one colony per construct into 100 mL liquid culture. Grow overnight at 37 C. |
*Harvest and prepare cells according to Qiagen [https://static.igem.org/mediawiki/2015/5/58/QIAGEN-Plasmid-Purification-Handbook--April-2012.pdf Maxiprep] protocol. | *Harvest and prepare cells according to Qiagen [https://static.igem.org/mediawiki/2015/5/58/QIAGEN-Plasmid-Purification-Handbook--April-2012.pdf Maxiprep] protocol. | ||
+ | |||
==BsaI Digestion== | ==BsaI Digestion== | ||
*Use 4 uL [https://www.neb.com/products/r0535-bsai NEB BsaI]in a 50 uL reaction to digest either: | *Use 4 uL [https://www.neb.com/products/r0535-bsai NEB BsaI]in a 50 uL reaction to digest either: |
Latest revision as of 06:39, 18 September 2015
Contents
MaSp ICA Fragments
- Iterative Capped Assembly (ICA) requires 50 ng of fragment for each extension cycle.
DNA Amplification
- Starting from fully sequenced plasmids, DNA can be amplified in two ways.
PCR Amplification
- Amplify 220 pg of each plasmid in a 50 uL reaction with VF2 and VR primers (the biobrick sequencing primers) and using Q5 polymerase and the GC enhancer. Set up 8x 50 uL reactions.
- Anneal at 63 C, using 18 cycles.
- Pool all 8 reactions together, and PCR purify
Maxiprep (Cloning Amplification)
- Streak the glycerol stocks on chloramphenicol plates, and grow overnight at 37 C.
- Pick one colony per construct into 100 mL liquid culture. Grow overnight at 37 C.
- Harvest and prepare cells according to Qiagen Maxiprep protocol.
BsaI Digestion
- Use 4 uL NEB BsaIin a 50 uL reaction to digest either:
- 1.0 to 1.5 ug of PCR amplified MaSp.
- 5 ug of whole plasmid.
- Digest at 50 C for 2 hrs, then heat kill at 65 C for 20 min.
- Run all of the digest mixture on 1.8% gel for purification.
- Excise 102 bp band, and use Qiagen Kit to gel extract.
- The extracted pieces can be used in ICA later on.