Team:Waterloo/Protocols
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- | + | <h3><b> Plasmid Isolation of S. epidermidis and S. aureus </b></h3> | |
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- Pellet cells from 2-10mL of overnight culture. | - Pellet cells from 2-10mL of overnight culture. | ||
- Centrifuge for 1 minute at 13000rpm. Discard supernatant. | - Centrifuge for 1 minute at 13000rpm. Discard supernatant. | ||
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- | Transformation Protocol of S. epidermidis/aureus | + | <h3><b>Transformation Protocol of S. epidermidis/aureus</b></h3> |
- Inocculate 10mL TSB (+25uL tetracycline if S. epidermidis) overnight culture (12-16 hours) | - Inocculate 10mL TSB (+25uL tetracycline if S. epidermidis) overnight culture (12-16 hours) | ||
- Dilute the overnight culture to an OD@578nm of ~0.5 in fresh prewarmed media, note the volume (x) | - Dilute the overnight culture to an OD@578nm of ~0.5 in fresh prewarmed media, note the volume (x) | ||
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- | sarA P1 Promoter Efficacy Assay: | + | <h3><b> sarA P1 Promoter Efficacy Assay: </b></h3> |
The sarA P1 promoter (BBa_K1323021) and a J23101 promoter were inserted in front of a GFP coding sequence (BBa_ E0240). The resulting two plasmid constructs (now a GFP cassette) were independently transformed into DH5α. A diagnostic restriction digest was run to confirm the insert composition within each of these two GFP cassettes. Colonies were streak purified and grown on LB supplemented with an appropriate antibiotic. Overnight cultures were then prepared in triplicate. 50 µL was then used to inoculate fresh media. When each media preparation reached an OD600 of 0.5-0.6, the GFP fluorescence was measured on a plate reader. Quantification of sarA P1 is therefore measured relative to the amount of YFP produced from the standard J23101 promoter. | The sarA P1 promoter (BBa_K1323021) and a J23101 promoter were inserted in front of a GFP coding sequence (BBa_ E0240). The resulting two plasmid constructs (now a GFP cassette) were independently transformed into DH5α. A diagnostic restriction digest was run to confirm the insert composition within each of these two GFP cassettes. Colonies were streak purified and grown on LB supplemented with an appropriate antibiotic. Overnight cultures were then prepared in triplicate. 50 µL was then used to inoculate fresh media. When each media preparation reached an OD600 of 0.5-0.6, the GFP fluorescence was measured on a plate reader. Quantification of sarA P1 is therefore measured relative to the amount of YFP produced from the standard J23101 promoter. | ||
- | sRNAi Silencing Assay: | + | <h3><b> sRNAi Silencing Assay:</b></h3> |
The YFP (BBa_K1323010) cassette was co-transformed with two sRNA constructs (BBa_K1323006, BBa_K1323007) into DH5α. Transformation of a YFP cassette on it’s own served as a positive control for YFP expression, while an sRNA cassette (BBa_K1323008) which targets a non-YFP sequence was the negative control. Colonies were streak purified and grown in LB supplemented with ampicillin. A diagnostic restriction digest was run to confirm that co-transformation was successful in the three co-transformed colonies. The digests were all done with EcoR1 and Pst1; in this way the inserts of each of the two co-transformed plasmids could be distinguished by gel electrophoresis. Overnight cultures were then prepared in triplicates. 50 µL of overnight culture was used to inoculate fresh media. When each media preparation reached an OD600 of 0.5-0.6, the YFP fluorescence was measured on a plate reader. | The YFP (BBa_K1323010) cassette was co-transformed with two sRNA constructs (BBa_K1323006, BBa_K1323007) into DH5α. Transformation of a YFP cassette on it’s own served as a positive control for YFP expression, while an sRNA cassette (BBa_K1323008) which targets a non-YFP sequence was the negative control. Colonies were streak purified and grown in LB supplemented with ampicillin. A diagnostic restriction digest was run to confirm that co-transformation was successful in the three co-transformed colonies. The digests were all done with EcoR1 and Pst1; in this way the inserts of each of the two co-transformed plasmids could be distinguished by gel electrophoresis. Overnight cultures were then prepared in triplicates. 50 µL of overnight culture was used to inoculate fresh media. When each media preparation reached an OD600 of 0.5-0.6, the YFP fluorescence was measured on a plate reader. | ||
- | + | <h3><b> Antibiotic Resistance Profile</b></h3> | |
- | Antibiotic Resistance Profile | + | |
The construct containing both chloramphenicol and erythromycin (pSB1CE3 shuttle vector) was electroporated into s.epidermidis. Three colonies were streak purified and diagnostic restriction digests were done to confirm that the insert was present. Overnight TSB cultures were supplemented with 10 ug/mL erythromycin, and the inoculation was done in triplicate. The negative control for this experiment is a stock S.epidermidis (ATCC12228). It lacks erythromycin resistance but has chloramphenicol resistance (S. epiderimidis has this resistance endogenously). 10 uL of the overnight cultures were used to inoculate fresh TSB that was supplemented with 10, 50, or 100 ug/mL of erythromycin and chloramphenicol. The negative growth control was 75 ug/mL of kanamycin and the positive growth control was TSB without antibiotic. The samples were incubated at 37 ͒C, shaking at 200 rpm. The OD600 was read after 6 hours of incubation. | The construct containing both chloramphenicol and erythromycin (pSB1CE3 shuttle vector) was electroporated into s.epidermidis. Three colonies were streak purified and diagnostic restriction digests were done to confirm that the insert was present. Overnight TSB cultures were supplemented with 10 ug/mL erythromycin, and the inoculation was done in triplicate. The negative control for this experiment is a stock S.epidermidis (ATCC12228). It lacks erythromycin resistance but has chloramphenicol resistance (S. epiderimidis has this resistance endogenously). 10 uL of the overnight cultures were used to inoculate fresh TSB that was supplemented with 10, 50, or 100 ug/mL of erythromycin and chloramphenicol. The negative growth control was 75 ug/mL of kanamycin and the positive growth control was TSB without antibiotic. The samples were incubated at 37 ͒C, shaking at 200 rpm. The OD600 was read after 6 hours of incubation. |
Revision as of 03:22, 18 October 2014