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Team:Technion/13 August 2012
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__NOTOC__ | __NOTOC__ | ||
==Ilya== | ==Ilya== | ||
| + | - PCR to get I0462 from F2620 failed. Probably due to the primer tail containing the RFC10 suffix which annealed to the suffix in the plasmid. This way the primer wasn't available to serve as the AS primer in the PCR reaction.<br> | ||
| + | - Repeated the PCR reaction with a gradient PCR and in GC buffer. Will check results tomorrow.<br> | ||
| + | - Put starters of the pSB3C5 and R0062+Fus1 in pSB1A2 for minipreps tomorrow. | ||
==Inbal== | ==Inbal== | ||
| - | + | -Transformation of the ligated products (pSB1C3+SP6) to 100ul component cells (TOP10 Rb bacteria). <br> | |
| + | -Recovery on SOB for 1 hr at 37C in the shaker. <br> | ||
| + | -Plating 100ul on LB+CM plate. <br> | ||
| + | -The rest of the SOB+cells were centrifuged for 1 min, max rpm, then I discarded the fluid. I plated the precipitation on LB+CM plate. | ||
| + | -I stored both plates in 37C incubator overnight. | ||
==Asaf== | ==Asaf== | ||
| Line 12: | Line 19: | ||
==Noa== | ==Noa== | ||
| - | PCR for xyIE and Alkaline Phosphatase. <br> | + | PCR for xyIE and Alkaline Phosphatase, gel electroporation and PCR purification. <br> |
| - | Starters for Chris's plasmids: 5 microL spectinomycin X 6 | + | Starters for Chris's plasmids: <br> |
| - | + | 5 microL spectinomycin + 5 ml LB X 6 -- '''NOT DONE''' <br> | |
| + | 13 microL chloromphenicol + 5 ml LB X 6 -- '''NOT DONE''' | ||
==Evgeni== | ==Evgeni== | ||
| + | Digestion and purification of: <br> | ||
| + | - pPROLar plasmid with cerulean reporter gene under pLac/Ara by HindIII and BamHI | ||
| + | - T7 PCR product by HindIII and NheI | ||
| + | |||
| + | After digestion, I had run pPROLAr on gel and got bands of expected size, as a control uncut plasmid had been used. The final product was purified from a gel by a kit. Nanodrop had showed really low concentration of final product - 5.6 ng/ul. I'll try to do another restriction and purification tomorrow. | ||
| + | T7 was purified by DNA purification kit, without using gel. | ||
==Shahar== | ==Shahar== | ||
Latest revision as of 13:35, 20 August 2012
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Ilya
- PCR to get I0462 from F2620 failed. Probably due to the primer tail containing the RFC10 suffix which annealed to the suffix in the plasmid. This way the primer wasn't available to serve as the AS primer in the PCR reaction.
- Repeated the PCR reaction with a gradient PCR and in GC buffer. Will check results tomorrow.
- Put starters of the pSB3C5 and R0062+Fus1 in pSB1A2 for minipreps tomorrow.
Inbal
-Transformation of the ligated products (pSB1C3+SP6) to 100ul component cells (TOP10 Rb bacteria).
-Recovery on SOB for 1 hr at 37C in the shaker.
-Plating 100ul on LB+CM plate.
-The rest of the SOB+cells were centrifuged for 1 min, max rpm, then I discarded the fluid. I plated the precipitation on LB+CM plate.
-I stored both plates in 37C incubator overnight.
Asaf
Hila
Lior
Noa
PCR for xyIE and Alkaline Phosphatase, gel electroporation and PCR purification.
Starters for Chris's plasmids:
5 microL spectinomycin + 5 ml LB X 6 -- NOT DONE
13 microL chloromphenicol + 5 ml LB X 6 -- NOT DONE
Evgeni
Digestion and purification of:
- pPROLar plasmid with cerulean reporter gene under pLac/Ara by HindIII and BamHI
- T7 PCR product by HindIII and NheI
After digestion, I had run pPROLAr on gel and got bands of expected size, as a control uncut plasmid had been used. The final product was purified from a gel by a kit. Nanodrop had showed really low concentration of final product - 5.6 ng/ul. I'll try to do another restriction and purification tomorrow.
T7 was purified by DNA purification kit, without using gel.
