IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/19

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Team Flavor DTL of Mira/Brazz+StrepII into STOP+V0120 Digestion Digestion Reactions Stop v0120 1 Stop v0120 2 DNA 10 8 FD Buffer (10x) 2 2 diH2O 6 8 Xba1 1 1 EcoRI 1 1 Ligation Reactions Mira N + Stop Mira C + Stop Brazz N + Stop Brazz C + Stop Stop only Control diH2O 3 3 11 6 14 T4 DNA ligase buffer (10x) 2 3 2 2 2 DNA Insert 11 11 3 8 0 DNA Backbone 3 3 3 3 3 T4 DNA ligase 1 1 1 1 1 Confirm of valencene PCR (failed) Re-do of Valencene PCR (failed) Team Allergy Got back sequencing results of GerS, BetS, PDK All three samples of GerS and PDK sent in matched their corresponding sequences Ran PCR of Friday's digested B21. The gel suggested a partial digest: we saw two bands of backbone where we should have seen only one: Sending 6 samples of amiRNA (LTP.5), 5 samples of amiRNA (LTP), 6 samples of amiRNA (Bet) Second digest of B21 appears to have been more successful: Digestion Reactions V0120 DNA ~2.1 ugrams FD Buffer (10x) 2 diH2O 20-rest Xba1 1 EcoRI 1 phosphatized backbone afterwards for 20 min after letting digestion run for an hour concentrations backbone: (5 copies each ~ 35 ng/uL) Bet S/A:  ; Bet2S/A:  ; GerS/A:  ; LTPS/A:  ; PME:  ; Pal: Ligation Reactions BetS/A Bet2S/A LTPS/A GerS/GerA Pme Pal diH2O 10-rest 10-rest 10-rest 10-rest 10-rest 10-rest T4 DNA ligase buffer (10x) 1 1 1 1 1 1 DNA Insert 3 times excess 3 times excess 3 times excess 3 times excess 3 times excess 3 times excess DNA Backbone ~50ng ~50ng ~50ng ~50ng ~50ng ~50ng T4 DNA ligase 1 1 1 1 1 1 Ligated and transformed allergen parts Pme/pal Team Fence 5xGal4 Promoter Annealing Product Ligation Made a 1 in 1000 dilution of the annealing reaction Nanodrop ng/μL 260/280 5xGalpt 1/1000 dilution 17.6 2.25 For a ligation reaction, we want: 50ng of backbone A 1:3 ratio of backbone:insert 3(ng of backbone / basepairs of backbone) = (ng of insert / basepairs of insert) 5xGalpt is 180 bases long, B21 backbone is 3.2kb. So we want 8.4375 ng of plasmid To reach 8.4ng of 5xGalpt, made a 1 in 2000 dilution of the 5xGalpt Annealing product Ligation Reaction: 1μL 1/2000 5xGalpt 2.5μL B21 Backbone 6.5μL DH2O 1μL Quick Ligase 10μL 2x Quick Ligase Buffer B21 Control Ligation: 2.5μL B21 Backbone 7.5μL DH2O 1μL Quick Ligase 10μL 2x Quick Ligase Buffer Incubated at room temp for 15 mins, put on ice and immediately transformed. Transformations of 5xGalpt, NLS.Serine anneal, and B21 Backbone control Thawed 3 Turbo chemically competent cells on ice Pippeted 1μL of the ligation or annealing reaction into its own tube of turbo cells Chilled on ice for 30 mins Heatshocked in 42°C water on heat block for 30 seconds Chilled on ice for 2 mins Added 170μL SOC medium to each tube, set to shake at 37°C for 15 mins Streaked on LB + amp plates, and left in incubator overnight