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

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DTL of Mira/Brazz+StrepII into STOP+V0120
Digestion

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:




 * 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:


 * Ligated and transformed
 * allergen parts
 * Pme/pal

5xGal4 Promoter Annealing Product Ligation

 * Made a 1 in 1000 dilution of the annealing reaction

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