August/7 August 2009

Today we carried out the following procedures (in rough chronological order):

1. Checked the cell cultures transformed and plated out yesterday (8/6) for colony formation and made an approximate count of the number of colonies.

(plate number)-(location on plate) (no. of colonies) ---            F16010  (2-24G)                        none M0100  (2-15F)                        none I742158 (1-19B)                    >50 B0034  (1-2M)                         >50 I0462  (1-8O)                         ~10 K118002 (2-16F)                    ~10 K118003 (2-16H)                    ~10 B0015 (1-23L)                         >50 I1466 (1-23J)                         ~10 C0077 (1-14F)                        ~10 C0076 (1-14D)                        ~10 K081020 (2-12H)                    none S03878 (2-16M)                      ~10 C1079  (2-8M)                         ~10

2. Conducted a 'miniprep' to harvest and check concentration of the EpsE (molecular clutch) plasmids from 3 test tube cultures incubated since yesterday (8/6). Result of Nanodrop concentration check:

(test tube no.) [260/280] [260/230] (concentration) ---      1           2.14      1.65     37.6 ng/ul 2          2.07      1.95     62.9 ng/ul 3          1.95      1.33     60.6 ng/ul

3. Prepared kanamycin antibiotic solution from kanamycin sulfate and MilliQ water. 100 mg kanamycin sulfate + 10 ml MilliQ water -> 10 ml of 10 mg/ml kanamycin solution.

4. Transformed compe[tent cells with the following parts to amplify them:

(plate number)-(location on plate) (antibiotic resistance) ---            F16010  (2-24G)                             A             K081020 (2-12H)                             K             K118013 (2-18F)                             A             R0071 (1-12C)                             A             I14017 (1-18L)                             K             R0062 (1-6O)                              A             R0079 (1-12A)                             A             R0077 (1-10K)                             A Note: 2-24G and 2-12H were transformed yesterday but produced no colonies. 2-18F was marked for transformation yesterday but 2-15F was mistakenly transformed instead.

5. Treated a sample of EpsE-containing plasmids with EcoRI and SpeI restriction endonucleases to isolate and check length of amplified EpsE part. The plasmid-buffer-endonuclease mixture was incubated and left to react overnight (Incubation at 37 degrees Celsius began at 16:00).

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