- Results from last week:
- 1 tube of LacI-RBS 34 was obtained!
- Cells with LacI-RFP plasmids in fact appeared in pink color. And the color looks basically the same as the cells with LacI-GFP that we plated dozens times before.
- Hung, Lu Chao: try ligation for LacI (vector)-GFP(insert), Fe(vector)-GFP(insert), E7(insert)-empty plasmid vector from GFP.
- 10-1040am: prepare double digestion mixtures for GFP vector (with EcoRI and PstI) , and 2 mixtures of GFP inserts (cut with XbaI and PstI). Incubate for 3 hours.
- 1120-1150: prepare double digestion mixtures for LacI vector (SpeI and PstI), and Fe vector (SpeI and PstI). Incubate for 2 hours.
- 1415: PCR purification for the above 5 mixtures.
- 1500-1600: gel loading and running.
- 1615-1700: gel extraction. After that, we used nanodrop to measure the concentrations and purities of the above samples. The results showed low concentrations and poor purities. A suggestion by Dr. Tan was to use new clean buffer for the gel before gel extraction. Also, because columns for Minelute PCR purification kit went out of stock, we decided to store the digested DNAs in -20 degrees fridge for Tuesday.
- Lu Chao:
- Transformation and cloning for new empty plasmids : BBa_I739201 (4kb) and BBa_I751000 (4.3kb).
- Inoculation for RBS 34, pLacI, pFe, GFP (3 tubes each).
- Gel check for E7 (E-X...S-P) PCR products.
- 1st gel run with 10ul was unsuccesful, 10ul causes extreme smearing and pseudo E7 (E-X...S-P) was at 1.2k bp band which was incorrect.
- 2nd Gel run with 2ul of E7 (E-X...S-P) PCR from the same tubes and with previously done E7 (X...S) as control. Both bands show a distinct though faint band at 2k mark.
- Conclusions are the amount of PCR product added matters.
- Experimentations with different amount on 8th July will take place, 2ul,4ul,6ul,8ul.
- 8th July will be creating stocks of purified E7 DNA.
- Ran PCR on E7 DNA for more gel runs tomorrow.
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