Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics/Protocols/MaSp ICA Fragments"

(→‎BsaI Digestion)
(→‎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 each colony into 100 mL liquid culture. 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 [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

iGEM UCLA




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.