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Well... quite a bit of work I'm not sure how far I get now, because I have to get to work ...

1. You would need a source for your DNA, a donor. By searching the ncbi for the key enzymes (you would need to search for all the different synonyms) and filter by organisms, which would be available in your lab.

2. Design primers - yeah thats quite a bit, I can not design you any primers with no ORF. Primer design is "simple as fuck" as I ve learned here in the Nexus. ... But for noobs in short: You need two primers. One forward, one reverse. For the forward you copypaste the lets say first twenty bp from the start point (AUG). Check for equal ammounts of g,c,t,a's. You would add in front of the sequence a restriction recognition site, lets say SacI (GAGCTC). Of course you must check for the SacI within the ORF, because when it is present, that would suck For beginners its wise to add 2-3 bp more, as polymerases are bastards. Calculate melting temp!
For reverse, copypaste last 20 bp form end, invert them, add other restriction site [e.g PstI(CTGCAG), check for restriction site presence within ORF, add few bp for good luck.

example: ORFX: auggcta....................gctagtg

fw: ct(GAGCTC)auggcta......

rev: CGATCACCTGCAGga

Sorry if Im talking bullshit, but as mentioned before Im in a hurry

2. Order primers

3. Isolate DNA

4. PCR: 50µl Pfu batch, 37x/mt°C-5°C/(1kb/1.2min)

5. Recover DNA over gelelectrophoresis

6. 20µl Taq batch witn dATPs, "a-tailing" 72°C, 15min

7. Ligate into subvector, e.g. pGEM-Teasy

8. Transform E.coli, let grow overnight

10. Screen via b/w and antibiotic resistance

11. Prepare 2mL cultures of different colonies, let grow over night, dont forget antibiotic

12. Miniprep

13. Control for positive plasmids via restriction double digest, one cut within ORF one in vector

14. If postive, excise ORF from subvector via SacI/PstI, recover fragment of interest via gelelectrophoresis

15. Ligate ORF in expressionvector

16. Transform E. coli. let grow overnight

17. Prepare 2mL cultures for overnight growth (antibiotics!)

18. MAXIprep or very good miniprep, as most expression vectors ar lowcopy plasmids

19. Check via restrictiondigest, when positive prepare glycostocks for permanent storage at -80°C

20. Prepare a 200ml culture with additional 50mg of l-tryptophane of positive colony. Let grow to an OD600 of about 0,5 and induce vector with IPTG and then let grow at 28°C overnight (antibiotics).

21. Extract similar to MHRB, but shorter

22. Run extract over TLC with a DMT reference

23. Run extract over GC-MS with a DMT ref.

[USER=769]@Infundibulum[/USER]: as mentioned, im in a hurry... off to work now.in this theoretical approach might be errors and you are welcome to correct and ask for further details - but need to wait couple of hours for the answer....hope it is not to exhautively? im not sure how much detail you were expecting

<blockquote data-quote="crystalizer" data-source="post: 3444644" data-attributes="member: 9193">Well... quite a bit of work <img src="data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7" class="smilie smilie--sprite smilie--sprite2" alt=";)" title="Wink&nbsp; &nbsp; ;)" loading="lazy" data-shortname=";)" /> I&#039;m not sure how far I get now, because I have to get to work ... 1. You would need a source for your DNA, a donor. By searching the ncbi for the key enzymes (you would need to search for all the different synonyms) and filter by organisms, which would be available in your lab. 2. Design primers - yeah thats quite a bit, I can not design you any primers with no ORF. Primer design is &quot;simple as fuck&quot; as I ve learned here in the Nexus. ... But for noobs in short: You need two primers. One forward, one reverse. For the forward you copypaste the lets say first twenty bp from the start point (AUG). Check for equal ammounts of g,c,t,a&#039;s. You would add in front of the sequence a restriction recognition site, lets say SacI (GAGCTC). Of course you must check for the SacI within the ORF, because when it is present, that would suck <img src="data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7" class="smilie smilie--sprite smilie--sprite2" alt=";)" title="Wink&nbsp; &nbsp; ;)" loading="lazy" data-shortname=";)" /> For beginners its wise to add 2-3 bp more, as polymerases are bastards. Calculate melting temp!For reverse, copypaste last 20 bp form end, invert them, add other restriction site [e.g PstI(CTGCAG), check for restriction site presence within ORF, add few bp for good luck. example: ORFX:&nbsp;&nbsp; auggcta....................gctagtgfw:&nbsp; &nbsp; ct(GAGCTC)auggcta......rev: CGATCACCTGCAGgaSorry if Im talking bullshit, but as mentioned before Im in a hurry <img src="data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7" class="smilie smilie--sprite smilie--sprite2" alt=";)" title="Wink&nbsp; &nbsp; ;)" loading="lazy" data-shortname=";)" />2. Order primers3. Isolate DNA4. PCR: 50µl Pfu batch, 37x/mt°C-5°C/(1kb/1.2min)5. Recover DNA over gelelectrophoresis6. 20µl Taq batch witn dATPs, &quot;a-tailing&quot; 72°C, 15min7. Ligate into subvector, e.g. pGEM-Teasy8. Transform E.coli, let grow overnight10. Screen via b/w and antibiotic resistance11. Prepare 2mL cultures of different colonies, let grow over night, dont forget antibiotic12. Miniprep13. Control for positive plasmids via restriction double digest, one cut within ORF one in vector 14. If postive, excise ORF from subvector via SacI/PstI, recover fragment of interest via gelelectrophoresis15. Ligate ORF in expressionvector16. Transform E. coli. let grow overnight17. Prepare 2mL cultures for overnight growth (antibiotics!)18. MAXIprep or very good miniprep, as most expression vectors ar lowcopy plasmids19. Check via restrictiondigest, when positive prepare glycostocks for permanent storage at -80°C20. Prepare a 200ml culture with additional 50mg of l-tryptophane of positive colony. Let grow to an OD600 of about 0,5 and induce vector with IPTG and then let grow at 28°C overnight (antibiotics).21. Extract similar to MHRB, but shorter22. Run extract over TLC with a DMT reference23. Run extract over GC-MS with a DMT ref.[USER=769]@Infundibulum[/USER]: as mentioned, im in a hurry... off to work now.in this theoretical approach might be errors and you are welcome to correct and ask for further details - but need to wait couple of hours for the answer....hope it is not to exhautively? im not sure how much detail you were expecting</blockquote>

[QUOTE="crystalizer, post: 3444644, member: 9193"] Well... quite a bit of work ;) I'm not sure how far I get now, because I have to get to work ... 1. You would need a source for your DNA, a donor. By searching the ncbi for the key enzymes (you would need to search for all the different synonyms) and filter by organisms, which would be available in your lab. 2. Design primers - yeah thats quite a bit, I can not design you any primers with no ORF. Primer design is "simple as fuck" as I ve learned here in the Nexus. ... But for noobs in short: You need two primers. One forward, one reverse. For the forward you copypaste the lets say first twenty bp from the start point (AUG). Check for equal ammounts of g,c,t,a's. You would add in front of the sequence a restriction recognition site, lets say SacI[color=green] (GAGCTC)[/color]. Of course you must check for the SacI within the ORF, because when it is present, that would suck ;) For beginners its wise to add [color=cyan]2-3 bp[/color] more, as polymerases are bastards. Calculate melting temp! For reverse, copypaste [color=darkred]last 20 bp[/color] form end, invert them, add other restriction site [e.g PstI[color=violet](CTGCAG)[/color], check for restriction site presence within ORF, add [color=cyan]few bp[/color] for good luck. example: ORFX: [color=orange]auggcta......[/color].....[color=darkred].........gctagtg[/color] fw: [color=cyan]ct[/color][color=green](GAGCTC)[/color][color=orange]auggcta......[/color] rev: [color=darkred]CGATCAC[/color][color=violet]CTGCAG[/color][color=cyan]ga[/color] Sorry if Im talking bullshit, but as mentioned before Im in a hurry ;) 2. Order primers 3. Isolate DNA 4. PCR: 50µl Pfu batch, 37x/mt°C-5°C/(1kb/1.2min) 5. Recover DNA over gelelectrophoresis 6. 20µl Taq batch witn dATPs, "a-tailing" 72°C, 15min 7. Ligate into subvector, e.g. pGEM-Teasy 8. Transform E.coli, let grow overnight 10. Screen via b/w and antibiotic resistance 11. Prepare 2mL cultures of different colonies, let grow over night, dont forget antibiotic 12. Miniprep 13. Control for positive plasmids via restriction double digest, one cut within ORF one in vector 14. If postive, excise ORF from subvector via SacI/PstI, recover fragment of interest via gelelectrophoresis 15. Ligate ORF in expressionvector 16. Transform E. coli. let grow overnight 17. Prepare 2mL cultures for overnight growth (antibiotics!) 18. MAXIprep or very good miniprep, as most expression vectors ar lowcopy plasmids 19. Check via restrictiondigest, when positive prepare glycostocks for permanent storage at -80°C 20. Prepare a 200ml culture with additional 50mg of l-tryptophane of positive colony. Let grow to an OD600 of about 0,5 and induce vector with IPTG and then let grow at 28°C overnight (antibiotics). 21. Extract similar to MHRB, but shorter 22. Run extract over TLC with a DMT reference 23. Run extract over GC-MS with a DMT ref. [USER=769]@Infundibulum[/USER]: as mentioned, im in a hurry... off to work now.in this theoretical approach might be errors and you are welcome to correct and ask for further details - but need to wait couple of hours for the answer....hope it is not to exhautively? im not sure how much detail you were expecting [/QUOTE]