IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/08/04

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Team Allergy

 * amiRNA fragment production appears to have worked at every Tm we tried (click images for details):


 * V9/V10
 * Diagnostic digest of our gel :

(Should see ~ 7kb band and an 840 band) Gel 1: Ladder, 9, 11c1, 11c2, ladder, 25c1, 28c1, 28c2, 36c1, 36c2, ladder Gel 2: Ladder, 37c1, 37 c2, 38c1, 38c2, ladder
 * Realized that we hadn't gel purified our inserts
 * Gel purification of inserts (entire ihpRNA parts)

PCR of VP16.3
60μL reaction:
 * 1.5 μL pfx polymerase
 * 3μL DNTPs
 * 18μL enhancer buffer
 * 1.5μL MgSO4
 * 3μL 1/10 dilution VP16.Hind.Fwd
 * 3μL 1/10 dilution VP16.Rev
 * 3μL 1/10 dilution of VP16 (1)
 * 12μL amp buffer
 * 5μL DH2O

Using program: 1=94°C for 2:00 2=94°C for :15 3=60/68°C for :30 4=68°C for :10 5=Goto 2, 30 times 6=4.0°C forever 7=end

Digestion of Gal-EcR
BamH1 and Nco1 12 digestions, one for each Gal-EcR miniprep Each:
 * 1μL BamH1
 * 1μL Nco1
 * 1μL 10x FD green buffer
 * 2-7μL EcR-Gal miniprep
 * 5-0μL DH2O, to bring total volume to 10μL

Ran a 1% gel of the digest



Confirmation Digests

 * Ran confirmation digests of miniprepped J45004-1, J45004-2, J45017-1, J45017-2
 * Digested approximately 500ng with xbaI/speI
 * Ran confirmation digests of miniprepped Miraculin N strep+stop and V24, Miraculin C strep+stop and V24, Brazzein N strep+stop and V24, Brazzein C strep+stop and V24.
 * Digested 400-500ng with notI/speI



Retry Ligating j45004 and v0120

 * Used PCR purified J45004 that had been previously digested with xbaI/pstI
 * Used v0120 from Team Vector that they had digested with xbaI/pstI and used in a successful ligation
 * 3:1 insert to backbone ration used in ligation
 * 52ng of insert and 50ng v0120 backbone
 * J45004 insert is 1100 bp
 * Transformed and plated on LB+Amp plates and left overnight


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