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Team:Gothenburg/Calendar
From 2014.igem.org
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The team received mandatory safety training and general lab routine information.
A really cool video about Chalmers' chemical safety routine (recorded at our very own lab!) can be seen in http://www.chalmers.se/insidan/EN/about-chalmers/environmental-work/policies/chemical-safety-routine |
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As a part of the mandatory training required by the SysBio group, the team received instructions on how to operate the centrifuges
and autoclaves of the labs.
We also worked on our risk declaration; a mandatory document containing our experiments descriptions and information about the chemicals we are going to use, their handling, storage and associated risk, as well as waste handling. |
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Our Risk Declaration was approved by the lab manager and our supervisors. We also sign a safety certificate stating that we are aware of the safety instructions and with that, completed the mandatory "check-in list for new incomers" of the lab. |
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On the first day of laboration, we prepared LB, YPD and SC-Ura/His/Trp-Leu and YPD media.
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Succesfully PCR amplified five out of the seven parts from yeast genome via a Phusion High-Fidelity DNA Polymerase.
The PCR program used was:
A diagnostic gel was performed and the result was as follows:
[[File: Bio-Rad 2014-06-25 15hr 34min.jpg|frameless|300px]] The purification of the succesfully amplified parts was done using a PCR Purification Kit from GenJet following the instructions
of the manufacture's manual but with no addition of isopropanol. |
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Plasmid Purification Performed plasmid purification on E. coli cells containing plasmids p413, p414, p415, p416, p2055 and a plasmid containig our Cas9 sequence
(pTPG1-dCas9-UPGP)via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by
water on the last step. Amplification and purification of partsPerformed a PCR amplification of the non-synthetic parts that failed on the day before and on the dCas9 sequence and the three fluorescent proteins
-Yellow, Cyano and Red (YFP, CFP and RFP, respectively). The PCR was done using the Phusion High-Fidelity DNA Polymerase (see protocol here) and the program used was:
A diagnostic gel was performed and the result is as follows: |
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Gel extractionExtracted the fluorecent proteins amplified on the day before with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step. PCR amplification and mergingPerformed a PCR to fuse the pDSE4, pHO and dCas9 parts together. The PCR was done using the Phusion High-Fidelity DNA Polymerase (see protocol here) and the program used was:
A diagnostic gel was performed and the result is as follows: The bands did not seem correct, so this PCR was repeated on the next day. |
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PCR amplification and mergingRepeated the PCR to fuse the pDSE4, pHO and dCas9 parts together. The PCR was done using the Phusion High-Fidelity DNA Polymerase (see protocol here) and the program used was:
A diagnostic gel was performed and the result is as follows: This did not seemed to work either. Plasmid restrictionCleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
(alkaline phosphatase) and up to 20 UL of deionized sterile water. This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC. |
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PCR amplification and mergingRepeated the PCR to fuse the pDSE4, pHO and dCas9 parts together.The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:
A diagnostic gel was performed, however the sizes of the resulting fragments did not match the expected. Plasmid restrictionCleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
(alkaline phosphatase) and up to 20 UL of deionized sterile water. This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC. Samples were loaded into a gelGreen, that showed p413, p415 and p416 approximately in the 5000bp size band, while p414 presented two bands, one around 4000bp and another at 2000bp. this indicates that the 5000bp band corresponds to the uncut plasmid, while the 4000bp and 2000bp are probably contaminations. |
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Plasmid restrictionCleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
(alkaline phosphatase) and up to 20 UL of deionized sterile water. This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC. PCR amplificationAmplified the parts pDSE4, cas9 and pHO. The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:
A diagnostic gel was performed, however the sizes of the resulting fragments did not match the expected. |
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PCR amplificationRepeated the amplification of the parts pDSE4, cas9 and pHO. The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was:
A diagnostic gel was performed and the sizes of the fragments matched for the pDSE4 and pHO, but not for the Cas9. Gel extractionExtracted the DNA fragments previously amplified with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step. Concentration determinationThe concentration of all the fragments obtained was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 8.5 ng of DNA per UL to 42ng/UL. |
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Plasmid purificationPerformed plasmid purification on E. coli cells containing plasmids p413, p414, p415, p416 and p2055 via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step. Each plasmid was purified in duplicates. Plasmid restrictionCleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
(alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared. This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC. A diagnostic gelGreen was performed and showed that the cleavage of plasmids p413, p415 and p416 was successful. Gel extractionExtracted the cleaved plasmids with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step. Concentration determinationThe concentration of the circular and linear plasmids obtained was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 6.55 ng of DNA per UL to 543.4ng/UL. |
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