- Gel purification of BOO14
- Determine concentrations of BOO14 and pSB1C3
- Restriction digest of pSB1C3 with EcoRI and PstI
- Check plates with transformants for colonies
- Perform colony PCR to confirm if ligation and transformation were successful
- Prepare a replica plate
- Set up of pSB1C3-BOO14 cultures for mini preps
- Set up PCR reaction with potentially contaminated agents
- Gel electrophoresis of PCR products to determine which component is contaminated with DNA
- Mini prep to isolate pSB1C3-BOO14
- Repeat gel electrophoresis of pSB1C3-BOO14 restriction digest
- Perform PCR to amlify CWB out of the ''B. subtilis'' genome
- Dephosphorylation of pSB1C3
- Set up ligation of pSB1C3 and BOO14 over night
- Transformation of ''E. coli'' with ligation product pSB1C3-BOO14
- Analysis of colony PCR using gel electrophoresis
- Meeting with the supervisors
- Repeat colony PCR with uncontaminated reagents
- Analysis of PCR product with gel electrophoresis
- Restriction digest with EcoRI and SpeI to confirm correct insert (BOO14)
- Gel electrophoresis to analyse restriction fragments
- Gel electrophoresis of the PCR product to confirm correct amplification
- Set up midi prep cultures of pSB1C3-BOO14
- We successfully purified BOO14 from the gel using the QIAquickÆ Gel Extraction Kit (250) but used 50µl of ddH2O instead of elusion buffer in the last step.
- Then we determined the relative concentrations of BOO14 and pSB1C3 by gel electrophoresis and comparing the intensity of the bands with the 1kb ladder.
- Having determined the rough concentration of our DNA we set up over night ligation of pSB1C3 with BOO14. We used two different ratios of pSB1C3 to BOO14: 0.5µl:3.5µl and 1µl:3µl. As the final concentration of the ligation product is very low, we will transform E. coli to be able to analyse bigger quantities of the vector at a later point.
- E. coli was transformed with the pSB1C3-BOO14 ligation using chemical competence and heat shock. We prepared plates with our transformants to be incubated overnight.
- We reorganized and updated our Lab-Page on the Wiki, including new tables, upload of pictures and result as well as user-interface optimisation.
- The plates with pSB1C3-BOO14 transformants had colonies growing on it.
- Some of these colonies were tested by colony PCR to confirm that BOO14 was in the vector.
- Gel electrophoresis of the PCR product indicated that our ligation and transformation were successful, however due to contamination, as demonstrated by our negative control, we will have to repeat the colony PCR on Thursday. We set up an additional PCR to test the individual components of our PCR reaction to find out which one is contaminated with DNA.
- We also set up cultures of pSB1C3-BOO14 for mini preps tomorrow.
- Analysis of the PCR components indicated that the Barns buffer was contaminated with DNA.
- We prepared mini preps of pSB1C3-BOO14 from the cultures set up yesterday.
- We then repeated the colony PCR and analysed the product with gel electrophoresis. We observed a band at 100bp, which indicates that the ligation was successful, and that BOO14 is in pSB1C3.
- We tried to confirm this with a restriction digest of the mini prepped plasmid with EcoRI and SpeI, however, analysis with gel electrophoresis showed there was only a very faint band on the gel at 100bp maybe because the gel had been run too long.
- We repeated the gel electrophoresis but reduced the time it ran for to 10 minutes as BOO14 is very short. However we still only observed a very faint band at 100bp, so we can't be completely sure that the ligation was successful.
- Therefore we carried out another restriction digest with AseI (which cuts within B0014) and NcoI (which cuts within pSB1C3).
- We also performed a PCR to amplify LytC cell wall binding domain (CWB) from the Bacillus genome.
- Analysis of the PCR product with gel electrophoresis showed that lytC had not been amlified properly. There we will do a series of different PCR reaction tomorrow to determine the optimal temperatures.
Sunday, 22nd Aug 2010
- Analysis of the restriction fragments from yesterdays restriction digest with gel electrophoresis showed that:
- The ligation definitely worked! The correct fragment sizes were observed on the gel.
- The primers in the PCR may have annealed nonspecifically, because the PCR product did not resolve properly in the gel.
- To prepare the ligated plasmid pSB1C3-BOO14 for midi prep tomorrow, overnight cultures were set up using 100ml LB (containing chloramphenicol), which was innoculated with cells from the original replica plates.
- A second PCR was set up with a gradient of increasing annealing temperatures, which would hopefully ensure specific binding of primers. (We used taq in this case, just to see which temperature gave us the best result)
- The gel of the PCR products showed that all 3 PCRs were successful. So we can now use Pfu in the next PCR to obtain the LytC cell wall binding domain.