Etchevers:Notebook/STRA6 in eye development/2009/03/23

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Minipreps of possible PAX6 expression vectors

Adeline has sequenced PAX6 and seen that both the long (minority) and short (majority) isoforms are present in her bands amplified with the primers.

She digested the inserts with Enzymes de restriction:

  • 5’: Not1
  • 3’: XhoI

Utilisation vecteur pcDNA 3.1/myc-his(+) C MCS : so that means if I linearize with EcoRI (and appropriate buffer) should be alright, because upstream of NotI (as would have been HindIII). Maybe, if there is an insert, will get multiple bands - I didn't think to check if there were EcoRI in the cDNA.

Regular primers should add in 1410 bp to the vector, which is 5.5 kb. Make sure your agarose gel is low density! If any are positive, should check that can double digest the XhoI+ and not the XhoI- clones - the latter is for run-on expression to make a fusion with the poly-His tag.

Used my regular miniprep protocol, made up fresh STET. Precipitated with 50% isopropanol, rinsed 70% EtOH before resuspension directly in

  • 44 μL H2O
  • 5 μL 10x NEB EcoRI buffer (from end of 2005!)
  • 1 μL EcoRI (NEB same lot).

1h at 37°C.

  • Heather 08:03, 23 March 2009 (EDT):

In fact, more like 2h. Then spun down for 15 minutes while poured a 0.7% agarose gel in TBE. Deposited 10 μL each + 2 μL dye. The gel must be >0.5% from evaporation because it's stiffish and things are migrating slowly. Apparently 2 bands in nearly every well (cut and uncut?) need to migrate more to see.

  • Heather 11:49, 23 March 2009 (EDT):