IGEM:metu/2009/Notebook/wound dressing/2009/08/14

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14.08.2009
1. Digestion of LuxR-RBS(05.08.09) has been done by PstI with following amounts,

- 2.71 microliters plasmid DNA - 1 microliter PstI - 2 microliter buffer - 14.29 microliters nuclease free water

However, the results weren't successful. The digested ones were in the same position with the control group.

2. The digestion will be performed firstly with XbaI, then with pstI this time. The digestion with XbaI was performed with two different samples from the same plasmid

- 2.65 microliters plasmid DNA - 1 microliter Fast Digest XbaI - 2 microliters Fast Digest Buffer - 14.35 microliters nuclease free water
 * The digestion of LuxR-RBS(11.08.09) was performed with following amounts;

- 2.71 microliters plasmid DNA - 1 microliter Fast Digest XbaI - 2 microliters Fast Digest Buffer - 14.29 microliters nuclease free water
 * The digestion of LuxR-RBS(05.08.09) was performed with following amounts;

3. After digestions, the purification and cleaning of plasmid have been done with PCR Purification kit from Fermentas. After the purification step, in 30 microliter elution buffer, the plasmid DNAs have been acquired.

4. The acquired plasmid DNA has been digested with Fast Digest PstI again with foolowing amounts; - 30 microliters plasmid DNA in elution buffer - 4 microliters Fast Digest Buffer - 2 microlites Fast Digest PstI enzyme - 4 microliters nuclease free water The results of electrophoresis were disappointing again. We couldn't observe two sided cut plasmid. Only one side of plasmids were cut.

5. We decided to use the conventional restriction enzymes because of inefficient cut of Fast Digest ones.

6. In 8 tubes, we placed different restriction enzymes (XbaI and PstI) from different years( The conventional restriction enzymes purchased this year and from previous years) with both LuxR-RBS plasmids. The added amounts are as follows;

1) New PstI has been added to LuxR-RBS from 11.08.09  - 2.65 plasmid DNA   - 1 microliter PstI   - 2 microliters Buffer O   - 41.35 microliters nuclease free water 2) Old PstI has been added to LuxR-RBS from 11.08.09 - 2.65 plasmid DNA - 1 microliter PstI - 2 microliters Buffer O  - 41.35 microliters nuclease free water 3) New PstI has been added to LuxR-RBS from 05.08.09  - 2.71 plasmid DNA   - 1 microliter PstI   - 2 microliters Buffer O   - 41.29 microliters nuclease free water 4) Old PstI has been added to LuxR-RBS from 11.08.09 - 2.71 plasmid DNA - 1 microliter PstI - 2 microliters Buffer O  - 41.29 microliters nuclease free water 5) New XbaI has been added to LuxR-RBS from 11.08.09  - 2.65 plasmid DNA   - 1 microliter XbaI   - 2 microliters Tango Buffer   - 14.35 microliters nuclease free water  6) Old XbaI has been added to LuxR-RBS from 11.08.09 - 2.65 plasmid DNA - 1 microliter XbaI - 2 microliters Tango Buffer - 14.35 microliters nuclease free water 7) New XbaI has been added to LuxR-RBS from 05.08.09  - 2.71 plasmid DNA   - 1 microliter XbaI   - 2 microliters Tango Buffer   - 14.29 microliters nuclease free water 8) Old XbaI has been added to LuxR-RBS from 05.08.09 - 2.71 plasmid DNA - 1 microliter XbaI - 2 microliters Tango Buffer - 14.29 microliters nuclease free water

The results of these digestions will be obtained tomorrow at 10 a.m and the accuracy of our conventional restriction enzymes will be determined.

7. For the new competent cell preparation, new seed TOP10 cells were used. The Top10 cells were added to 20 ml Luria-Bertani Broth


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