IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/08/06

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Team Allergy
amiRNA
 * amiRNA-transformed turbo cells didn't grow in YEB medium. There should be more LB+Amp available by the end of the day. In the meantime, we're digesting some more backbone (8x20ul X+P+B21)
 * ligations of Bet/LTP into V0120

ihpRNA
 * no colonies on plates from transformations done yesterday after 16 hours (we let these plates grow for longer)
 * redoing ligations of ihpRNA inserts in V9/V10
 * Redigested and gel purified some backbone

Concentrations: V9 (5 ng/uL); V10 (6 ng/uL)


 * 12 ligations each for V9 and V10

Confirmation Digest of J45004

 * J45004 in V0120 backbone; digested ~500ng DNA with Xba1/Pst1

Note: both gels images are the same, but with different contrasts/resolutions


 * 1) 1 kb Ladder (Invitrogen)
 * 2) 004-1
 * 3) 004-2
 * 4) 004-3
 * 5) Ladder

Backbone: 3.2 kb; Insert: 1.1 kb

Analysis
Both the backbone and the insert in lane 1 look like the correct length (3.2 and 1.1 kb respectively). Lane two consists of a backbone of the correct length, but the insert appears to be ~1.5 kb, too long for the J45004 BioBrick part. Lane 3 appears undigested; we are uncertain as to why.

Transformation of Mira/Brazz StrepII constucts into E45
Transformations: Mira N1 Mira N2 Mira C1 Mira C2 Brazz N1 Brazz N2 Brazz C1 Brazz C2
 * 100-200 ng of construct DNA was transformed with 15μL of E45 expression E. Coli; transformation procedure was followed; transformed cells were plated on LB+AMP and left to grow O/N

Digestions
pACT2
 * 1μL Bam
 * 1μL Xho
 * 6μL pACT2 (tube with a green top)
 * 1μL FD Green buffer
 * 1μL DH2O

RXRLc
 * 1μL Bam
 * 1μL Xho
 * 9μL RXRLc4
 * 2μL FD Green buffer
 * 7μL DH2O

Barstar
 * 1μL Eco
 * 1μL Pst
 * 2μL Barstar 3
 * 1μL FD Green buffer
 * 5μL DH2O

Barnase
 * 1μL Eco
 * 1μL Pst
 * 1.5μL Barnase 2
 * 1μL FD Green buffer
 * 5.5μL DH2O

5xGalUAS
 * 1μL Spe
 * 1μL Pst
 * 2μL 5xGalUAS (from Karmella)
 * 1μL FD Green buffer
 * 5μL DH2O

pENT Cup
 * 1μL Spe
 * 1μL Pst
 * 1μL pENT Cup
 * 1μL FD Green buffer
 * 6μL DH2O

EcR-Gal
 * 1μL Xba
 * 1μL Pst
 * 2μL EcR-Gal 7
 * 1μL FD Green buffer
 * 5μL DH2O

VP16
 * 1μL Spe
 * 1μL Pst
 * 5μL VP16(1)
 * 1μL FD Green buffer
 * 2μL DH2O

RXRLc
 * 1μL Xba
 * 1μL Pst
 * 9μL RXRLc4
 * 2μL FD Green buffer
 * 7μL DH2O

V24
 * 1μL Eco
 * 1μL Pst
 * 3μL V24
 * 1μL FD Green buffer
 * 4μL DH2O

V26
 * 1μL Eco
 * 1μL Pst
 * 9μL V26
 * 12μL FD Green buffer
 * 7μL DH2O

PhyB
 * 1μL Bam
 * 1μL Nco
 * 16μL PhyB
 * 2μL FD Green buffer


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