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Project:Public Biobrick: Difference between revisions
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=== Amplification of plasmid backbone (Hackspace) === | === Amplification of plasmid backbone (Hackspace) === | ||
The plasmid backbone is the vehicle for the biobrick, and is the means by which it will get into the competent cells. The procedure is in the same link as for competent cells. We did two versions of this reaction, one with the mastermix supplied by UCL, and one with the taq readymix that we use at the hackspace. We saw a band for the readymix one, but not for the mastermix one, so proceeded with the readymix one. | The plasmid backbone is the vehicle for the biobrick, and is the means by which it will get into the competent cells. It is a linearised plasmid backbone containing chlorophenicol resistance. The procedure is in the same link as for competent cells. We did two versions of this reaction, one with the mastermix supplied by UCL, and one with the taq readymix that we use at the hackspace. We saw a band for the readymix one, but not for the mastermix one, so proceeded with the readymix one. | ||
=== DNA extraction from O. Indolifex and PCR of two genes === | === DNA extraction from O. Indolifex and PCR of two genes === | ||
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*5: ANF 2 PCR product. | *5: ANF 2 PCR product. | ||
*6: ANF 3 PCR product. | *6: ANF 3 PCR product. | ||
*7: | *7: PSB1C3 PCR product. | ||
So we got no bands from the O. indofilex gDNA reactions, but we did get bands at the right position (285 bp) from 2/3 of the ANF template ones. ANF templates were ANF genes that had been previously PCRed with the non prefix/suffix primers. We also got a band from the positive control at the right position (2070bp). | So we got no bands from the O. indofilex gDNA reactions, but we did get bands at the right position (285 bp) from 2/3 of the ANF template ones. ANF templates were ANF genes that had been previously PCRed with the non prefix/suffix primers. We also got a band from the positive control at the right position (2070bp). | ||
=== Restriction digest | === Restriction digest and ligation === | ||
This step involves the insertion of the gene (ANF and MR in our case) into the plasmid backbone (PSB1C3). First the plasmid and the gene are incubated in separate reactions with restriction enzymes, to cut them at the appropriate parts so they can be ligated together. | |||
====Restriction digest and ligation 12/9==== | |||
=====Restriction digest===== | |||
Required mass of DNA in each reaction is 250ng, so quantities of PSB1C3 (50.9ng/ul) and ANF (19.2ng/ul) are calculated with this in mind. EcoR1 and PST1 are the restriction enzymes. BSA prevents adhesion of the enzyme(s) to reaction tubes and pipette surfaces, and stabilizes some proteins during incubation. NEB2 is a buffer for the restriction enzymes. | |||
'''Plasmid backbone''' | |||
* Total volume 25ul | |||
* PSB1C - 4.9ul | |||
* EcoR1 - 0.5ul | |||
* PST1 - 0.5ul | |||
* BSA - 0.3ul | |||
* dH20 - 16.3ul | |||
* NEB2 - 2.5ul | |||
'''ANF''' | |||
* Total volume 25ul | |||
* ANF - 13ul | |||
* EcoR1 - 0.5ul | |||
* PST1 - 0.5ul | |||
* BSA - 0.3ul | |||
* dH20 - 8.2ul | |||
* NEB2 - 2.5ul | |||
'''Control for ligation''' | |||
* Total volume 25ul | |||
* PSB1C - 4.9ul | |||
* PST1 - 0.5ul | |||
* BSA - 0.3ul | |||
* dH20 - 16.8ul | |||
* NEB2 - 2.5ul | |||
Incubated at 37C 30mins, 80C 20mins. | |||
=== Miniprep: Plasmid DNA Extraction === | === Miniprep: Plasmid DNA Extraction === | ||
TBA | TBA | ||
