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Team:Hong Kong HKUST/notebook/mod3
From 2013.igem.org
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<a href='#' class='head'>Week 2</a> | <a href='#' class='head'>Week 2</a> | ||
<div class='content' align="left"> | <div class='content' align="left"> | ||
| + | <p> </p> | ||
| + | <p><strong>08-07-2013</strong> <br /> | ||
| + | Result of MLS Sanger sequencing showed that DNA is not enough, the sequencing failed <br /> | ||
| + | Inoculation of MLS.PSB1C3 <br /> | ||
| + | Plasmid extraction of pEGFP-N1 <br /> | ||
| + | Digestion of pEGFP-N1 with NotI-HF & PstI-HF <br /> | ||
| + | Run gel, and it turned out that NotI-HF did not work </p> | ||
| + | <p> </p> | ||
| + | <p><strong>09-07-2013</strong> <br /> | ||
| + | Plasmid extraction of MLS biobrick <br /> | ||
| + | Digestion check on MLS.pSB1C3 product with EcoRI & PstI <br /> | ||
| + | Sanger sequencing of MLS biobrick <br /> | ||
| + | pEGFP-N1 digestion with PstI & NotI </p> | ||
| + | <p> </p> | ||
| + | <p><strong>10-07-2013</strong> <br /> | ||
| + | pcmv.myc.mito & pEGFP-N1 ligation, and transform into SURE on AMP plate <br /> | ||
| + | MLS biobrick Sanger sequencing result showed there is no pellet, repeat Sanger sequencing <br /> | ||
| + | Run gel for digestion check of MLS.psb1c3 with EcoRI & PstI </p> | ||
| + | <p> </p> | ||
| + | <p><strong>11-07-2013</strong> <br /> | ||
| + | Inoculation of pcmv.myc.mito.EGFP <br /> | ||
| + | MLS biobrick Sanger sequencing (Cont.) </p> | ||
| + | <p> </p> | ||
| + | <p><strong>12-07-2013</strong> <br /> | ||
| + | Plasmid extraction of pcmv/myc/mito.EGFP <br /> | ||
| + | pcmv/myc/mito.EGFP transformation check by digesting with PstI & NotI</p> | ||
| + | <p>Spread plate for confirmed colony </p> | ||
| + | <p> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
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<div> | <div> | ||
<a href='#' class='head'>Week 3</a> | <a href='#' class='head'>Week 3</a> | ||
| - | <div class='content' align="left"> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>15/7/2013</strong><br /> | ||
| + | pcmv/myc/mito.GFP inoculation</p> | ||
| + | <p> </p> | ||
| + | <p><strong>16/7/2013</strong><br /> | ||
| + | Plasmid Extraction of pcmv/myc/mito.GFP<br /> | ||
| + | Nanodrop: concentration is not high enough, will repeat inoculation</p> | ||
| + | <p> </p> | ||
| + | <p><strong>17-7-2013</strong><br /> | ||
| + | Inoculate 4 tubes of pcmv/myc/mito.GFP</p> | ||
| + | <p> </p> | ||
| + | <p><strong>18-7-2013</strong><br /> | ||
| + | pcmv/myc/mito.GFP plasmid extraction<br /> | ||
| + | PCR clone MLS (RFC25)</p> | ||
| + | <p> </p> | ||
| + | <p><strong>19-7-2013</strong><br /> | ||
| + | Run gel for MLS(25) PCR product, PCR clean up<br /> | ||
| + | Digestion of the MLS PCR product using EcoRI and PstI, gel check<br /> | ||
| + | Digestion of pSB1C3 with EcoRI & PstI, run gel</p> | ||
| + | <p> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
| Line 386: | Line 434: | ||
<div> | <div> | ||
<a href='#' class='head'>Week 4</a> | <a href='#' class='head'>Week 4</a> | ||
| - | <div class='content' align="left"> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>22-7-2013</strong><br /> | ||
| + | Gel purification of pSB1C3<br /> | ||
| + | Gel check of pSB1C3: band is too pale, repeat inoculation<br /> | ||
| + | Inoculation of pSB1C3.J04450<br /> | ||
| + | Repeat PCR of MLS(25)<br /> | ||
| + | Gel check of PCR product and PCR clean up<br /> | ||
| + | Digestion of MLS(25) with EcoRI & SpeI, run gel</p> | ||
| + | <p> </p> | ||
| + | <p><strong>23-7-2013<br /> | ||
| + | </strong>MLS(25) Gel purification (Cont), and gel check<br /> | ||
| + | pSB1C3 plasmid extraction<br /> | ||
| + | Digestion of pSB1C3 with EcoRI & SpeI</p> | ||
| + | <p> </p> | ||
| + | <p><strong>24-7-2013</strong><br /> | ||
| + | pSB1C3 Gel purification (Cont.)<br /> | ||
| + | MLS(25) & pSB1C3 ligation, and transform to CAM plate</p> | ||
| + | <p> </p> | ||
| + | <p><strong>25-7-2013</strong><br /> | ||
| + | Colony PCR of MLS.pSB1c3(25)<br /> | ||
| + | Inoculation of MLS.pSB1c3(25)</p> | ||
| + | <p> </p> | ||
| + | <p><strong>26-7-2013</strong><br /> | ||
| + | Plasmid extraction of MLS.pSB1c3(25)<br /> | ||
| + | Digestion of ligated MLS-pSB1C3 with EcoRI & SpeI, gel check<br /> | ||
| + | Gel check for MLS.pSB1c3(25) colony PCR product<br /> | ||
| + | Spread the remaining re-suspend of correct colony on CAM plate</p> | ||
| + | <p> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 18:37, 19 September 2013
June 2013
24-6-2013
Inoculation of pcmv/myc/mito
25-6-2013
Plasmid extraction for pcmv/myc/mito
Digestion of pcmv plasmid extraction product with PstI-HF and NotI-HF
Gel electrophoresis of pcmv digested product
Gel check for pcmv digestion product
26-6-2013
Gel purification of pcmv gel product
Gel check of pcmv gel purified product
PCR clone MLS from pcmv/myc/mito
Transformation of pEGFP-N1 with SURE
27-6-2013
Gel check for the MLS PCR product and PCR Clean Up
Digestion of MLS clean up product with EcoRI & PstI-HF
Inoculation of pEGFP-N1
28-6-2013
PCR Clean up of digested MLS
Plasmid Extraction of pEGFP-N1
Digestion of pEGFP-N1 with PstI-HF & NotI-HF
July 2013
02-07-2013
Gel Check of pEGFP-N1 plasmid extraction product to confirm presence of DNA
Digestion of pEGFP-N1 (with NotI-HF and PstI-HF) 2nd EGFP product:repeat digestion
Inoculation of pEGFP-N1 3rd EGFP: repeat Inoculation
Get pSB1C3.J04450 plasmid extraction product from Bida
Digestion of pSB1C3 with EcoRI and PstI-HF
03-07-2013
Gel electrophoresis of digested EGFP (2nd)
Plasmid extraction of pEGFP-N1 (3rd)
Digetion of pEGFP-N1 (3rd) using NotI-HF and PstI-HF
Ligation of MLS with pSB1C3
Transformation of ligated pSB1C3-MLS
04-07-2013
Gel purification of pEGFP-N1 (2nd) (cont.)
Gel analysis and gel electrophoresis of pEGFP-N1 (3rd)
Run gel for remaining 3rd EGFP (digested)
Ethanol precipitation of pEGFP-N1 (4th) (using remaining 3rd EGFP plasmid)
Digestion of pEGFP-N1 (4th) and gel electrophoresis
Transformation of pEGFP-N1 into DH10B
Colony PCR of MLS biobrick and gel electrophoresis
Inoculation of MLS colony no.2 & spread the remaining re-suspend colony 2 on CAM plate
05-07-2013
pEGFP-N1 troubleshoot
Digestion by PstI-HF, NotI-HF, PstI-HF +NotI-HF and gel check
Inoculate EGFP from transformed new competent cell
Plasmid extraction of MLS Biobrick
Sanger sequencing of MLS
08-07-2013
Result of MLS Sanger sequencing showed that DNA is not enough, the sequencing failed
Inoculation of MLS.PSB1C3
Plasmid extraction of pEGFP-N1
Digestion of pEGFP-N1 with NotI-HF & PstI-HF
Run gel, and it turned out that NotI-HF did not work
09-07-2013
Plasmid extraction of MLS biobrick
Digestion check on MLS.pSB1C3 product with EcoRI & PstI
Sanger sequencing of MLS biobrick
pEGFP-N1 digestion with PstI & NotI
10-07-2013
pcmv.myc.mito & pEGFP-N1 ligation, and transform into SURE on AMP plate
MLS biobrick Sanger sequencing result showed there is no pellet, repeat Sanger sequencing
Run gel for digestion check of MLS.psb1c3 with EcoRI & PstI
11-07-2013
Inoculation of pcmv.myc.mito.EGFP
MLS biobrick Sanger sequencing (Cont.)
12-07-2013
Plasmid extraction of pcmv/myc/mito.EGFP
pcmv/myc/mito.EGFP transformation check by digesting with PstI & NotI
Spread plate for confirmed colony
15/7/2013
pcmv/myc/mito.GFP inoculation
16/7/2013
Plasmid Extraction of pcmv/myc/mito.GFP
Nanodrop: concentration is not high enough, will repeat inoculation
17-7-2013
Inoculate 4 tubes of pcmv/myc/mito.GFP
18-7-2013
pcmv/myc/mito.GFP plasmid extraction
PCR clone MLS (RFC25)
19-7-2013
Run gel for MLS(25) PCR product, PCR clean up
Digestion of the MLS PCR product using EcoRI and PstI, gel check
Digestion of pSB1C3 with EcoRI & PstI, run gel
22-7-2013
Gel purification of pSB1C3
Gel check of pSB1C3: band is too pale, repeat inoculation
Inoculation of pSB1C3.J04450
Repeat PCR of MLS(25)
Gel check of PCR product and PCR clean up
Digestion of MLS(25) with EcoRI & SpeI, run gel
23-7-2013
MLS(25) Gel purification (Cont), and gel check
pSB1C3 plasmid extraction
Digestion of pSB1C3 with EcoRI & SpeI
24-7-2013
pSB1C3 Gel purification (Cont.)
MLS(25) & pSB1C3 ligation, and transform to CAM plate
25-7-2013
Colony PCR of MLS.pSB1c3(25)
Inoculation of MLS.pSB1c3(25)
26-7-2013
Plasmid extraction of MLS.pSB1c3(25)
Digestion of ligated MLS-pSB1C3 with EcoRI & SpeI, gel check
Gel check for MLS.pSB1c3(25) colony PCR product
Spread the remaining re-suspend of correct colony on CAM plate
