IGEM:metu/2009/Notebook/wound dressing/2009/08/19

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19.08.2009
1. Transformation result : We saw very few on plates. (we think because of compotent Top 10 cells transformation wasn't succesful) 2. Isolation of Rbs, AI2-ind,LUXR-Rbs, TRH, pEGF-Lard1, pRGF-HiA and storages of pEGF-Lard1, pRGF-HiA were made.

3. CaCl2 and LB were prepared.

4. We made (fast) monodigestion for GFP, EGF4, BB11 than purification and again (fast) monodigestion. Just BB11 was cutted. Then we made gell extraction. We couldn’t be sure for GFP and EGF4. 5. Double digestions of GFP(digested), EGF4 (undigested)were made.

For EGF3,        Prefix             Suffix GFP              EcoR1              Spe1 EGF4             Xba1               Pst1 BB11             EcoR1              Pst1 The following amounts were added to microtubes; GFP(28.07.09), monodigestion(fast): water 14.9 µl Buffer 2 µl DNA    2.1 µl EcoR1  1 µl =20 µl

EGF4(06.08.09), monodigestion(fast): water 14.5 µl Buffer 2 µl DNA    2.5 µl Xba1   1 µl =20 µl

BB11, monodigestion(fast): water 11.8 µl Buffer 2 µl DNA    5.2 µl Ecor1  1 µl =20 µl After PCR purification; For GFP, 28 µl solution 2 µl Spe1 4 µl buffer 6 µl water    =40 µl

For EGF4, 28 µl solution 2 µl Pst1 4 µl buffer 6 µl water    =40 µl

For BB11, 28 µl solution 2 µl Pst1 4 µl buffer 6 µl water    =40 µl

6. Electrophoresis order: 1)Leader / GFP control / GFP / EGF4 control / EGF4 / BB11 control / BB11 / Leader	  2)Leader / GFP / GFP / EGF4  / EGF4 / BB11 / BB11 / Leader 3)Leader / GFP / GFP / GFP control / Leader / EGF control / EGF / EGF 7. Mono digestions ( 16 hours incubation );      EGF4                     1/06.08.09                          2/11.08.09            water                14.48 µl                            12.87 µl            Buffer Tango          2 µl                                2 µl            DNA                   2.52 µl                             4.13 µl            Xba1                  1 µl       = 20 µl                  1 µl             =20 µl       GFP                      1/24.07.09                           2/28.07.09            water                13.43 µl                             12.87 µl            Buffer EcoR1          2 µl                                 2 µl            DNA                   3.57 µl                              2.06 µl            EcoR1                 1 µl      = 20 µl                    1 µl             =20 µl