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Project:Public Biobrick: Difference between revisions
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*INFO TO COME - which PCR program we used for the previous successful amplification of PSB1C3 at the hackspace? | *INFO TO COME - which PCR program we used for the previous successful amplification of PSB1C3 at the hackspace? | ||
Procedure: | =====Procedure:===== | ||
* ANF template reaction mix: 2ul of template, 2.5ul FP (ANFR1), 2.5ul RP (1RFNA), 25ul of taq readymix, 18ul dH20 | * ANF template reaction mix: 2ul of template, 2.5ul FP (ANFR1), 2.5ul RP (1RFNA), 25ul of taq readymix, 18ul dH20 | ||
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Both extractions showed up as faint bands in the wells, indicating gDNA extraction succeeded. Bands were same strength as I had previously seen for gDNA extractions at the hackspace, but should have been stronger. This could either be due to the extraction method not extracting enough DNA, or our visualisation technique being inadequate. | Both extractions showed up as faint bands in the wells, indicating gDNA extraction succeeded. Bands were same strength as I had previously seen for gDNA extractions at the hackspace, but should have been stronger. This could either be due to the extraction method not extracting enough DNA, or our visualisation technique being inadequate. | ||
Procedure: | =====Procedure:===== | ||
* gDNA extraction from the attachment to [https://groups.google.com/forum/?fromgroups=#!searchin/london-biohackers/alex/london-biohackers/osYVBsnCFvk/T2MhRlYdcI8J this] email, without the phenol/chloroform steps. Of the options in the protocol, we froze at -20C overnight, and extended the centrifuge steps by 10 mins each due to our less powerful centrifuge. Another difference was that after washing with 70% EtOh, pellet was large and off-white, indicating cellular debris. See [http://wiki.london.hackspace.org.uk/w/images/e/ef/Pellet_after_precipitation_and_washing.jpg photo]. So resuspended pellet in 30ul dH20, and discarded everything that did not dissolve. Procedure had previously worked at UCL. | * gDNA extraction from the attachment to [https://groups.google.com/forum/?fromgroups=#!searchin/london-biohackers/alex/london-biohackers/osYVBsnCFvk/T2MhRlYdcI8J this] email, without the phenol/chloroform steps. Of the options in the protocol, we froze at -20C overnight, and extended the centrifuge steps by 10 mins each due to our less powerful centrifuge. Another difference was that after washing with 70% EtOh, pellet was large and off-white, indicating cellular debris. See [http://wiki.london.hackspace.org.uk/w/images/e/ef/Pellet_after_precipitation_and_washing.jpg photo]. So resuspended pellet in 30ul dH20, and discarded everything that did not dissolve. Procedure had previously worked at UCL. | ||
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[[File:Gel_run_13_sept.JPG|left|400px|thumb|none|Gel run 13th sept]] | [[File:Gel_run_13_sept.JPG|left|400px|thumb|none|Gel run 13th sept]] | ||
Lanes from right: | =====Lanes from right:===== | ||
*1: Ladder. Note that loading dye is in the middle of ladder. Possibly because current turned off for 5 mins during gel run? | *1: Ladder. Note that loading dye is in the middle of ladder. Possibly because current turned off for 5 mins during gel run? | ||
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*4: O. Indofilex gDNA sample 1 (band just visible) | *4: O. Indofilex gDNA sample 1 (band just visible) | ||
*5: O. Indofilex gDNA sample 1 (band just visible) | *5: O. Indofilex gDNA sample 1 (band just visible) | ||
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=====PCR:===== | =====PCR:===== | ||
'''''We do not know whether this program was actually ran as it is written below, but here are the instructions.''''' | '''''We do not know yet whether this program was actually ran as it is written below, but here are the instructions.''''' | ||
* Initial D: 95C 30s | * Initial D: 95C 30s | ||
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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. (Not sure yet whether this means PCR succeeded, as 'ANF template' may already have been this size. Will explain what ANF template means when I understand it better myself!). 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. (Not sure yet whether this means PCR succeeded, as 'ANF template' may already have been this size. Will explain what ANF template means when I understand it better myself!). We also got a band from the positive control at the right position (2070bp). | ||
