Arking:JCAOligoTutoria21 - Revision history

JCAnderson: /* Gene Synthesis */

2013-01-29T18:11:11Z

Gene Synthesis

←Older revision		Revision as of 18:11, 29 January 2013
Line 4:		Line 4:

	===Gene Synthesis===		===Gene Synthesis===
-	Sometimes you don't have the source DNA. Perhaps the sequence comes from a source organism that is not available to you. Perhaps it is a eukaryotic CDS and you wish to express it in a bacteria. In that case, you might want to change the codon usage and eliminate all the intron sequences. If you have a sequence that is over 120bp or so, and you can't simply amplify the sequence from some source DNA, your only option is Gene Synthesis. There are a number of commercial suppliers who will synthesize your part and send you plasmid DNA (but it's pretty expensive). You can also do gene synthesis on your own. There is a website called GeneDesign http://~~genedesign~~.~~thruhere~~.~~net~~/cgi-bin/gd/gdRevTrans.cg that will help you design your part and determine the oligos you should order to assemble your part.	+	Sometimes you don't have the source DNA. Perhaps the sequence comes from a source organism that is not available to you. Perhaps it is a eukaryotic CDS and you wish to express it in a bacteria. In that case, you might want to change the codon usage and eliminate all the intron sequences. If you have a sequence that is over 120bp or so, and you can't simply amplify the sequence from some source DNA, your only option is Gene Synthesis. There are a number of commercial suppliers who will synthesize your part and send you plasmid DNA (but it's pretty expensive). You can also do gene synthesis on your own. There is a website called GeneDesign http://54.235.254.95/cgi-bin/gd/gdRevTrans.cgi that will help you design your part and determine the oligos you should order to assemble your part.

	===Overlap Extension===		===Overlap Extension===

JCAnderson: /* Enzymatic Inverse PCR (EIPCR) */

2012-01-26T16:02:46Z

Enzymatic Inverse PCR (EIPCR)

←Older revision		Revision as of 16:02, 26 January 2012
Line 47:		Line 47:
	CCAATAGATCTcatgaattccagaaatc		CCAATAGATCTcatgaattccagaaatc
	Last step: draw up the construction file and simulate it in ApE to make sure it will work:		Last step: draw up the construction file and simulate it in ApE to make sure it will work:
-	EIPCR ca9941/ca1168R on pBca9145-Bca1144#5 (2108 bp, BglII)	+	EIPCR ca9941/ca1168R on pBca9145-Bca1144#5 (2108 bp, eipcr)
-	~~Product is~~ pBca9145-Bca9941 ~~{A-20}~~	+	Digest eipcr (BglII, L, pcrdig)
		+	Ligate pcrdig (pBca9145-Bca9941)
	----		----
-	ca9941 EIPCR construction of 20 A's part	+	>ca9941 EIPCR construction of 20 A's part
	CCATAagatctAAAAAAAAAAAAAAAAAAAAggatcctaaCTCGAGctgcag		CCATAagatctAAAAAAAAAAAAAAAAAAAAggatcctaaCTCGAGctgcag
-	ca1168R Reverse BglII oligo for His6 EIPCR	+	>ca1168R Reverse BglII oligo for His6 EIPCR
	CCAATAGATCTcatgaattccagaaatc		CCAATAGATCTcatgaattccagaaatc

JCAnderson: /* Overlap Extension */

2012-01-26T16:00:22Z

Overlap Extension

←Older revision		Revision as of 16:00, 26 January 2012
Line 22:		Line 22:
	CTGATggatccTGGTGGAGCTATGCGGGATCGAACCGCAGACCTCCTGCGTTAGAAGCAGGCGCTC		CTGATggatccTGGTGGAGCTATGCGGGATCGAACCGCAGACCTCCTGCGTTAGAAGCAGGCGCTC
	That's it! Just write up the construction file and you are done. Note that I've pasted the part into a slightly different vector. The map of pBca9145-Bca1144#5 is [[Media:JCA_pBca9145-Bca1144.seq \| here]]:		That's it! Just write up the construction file and you are done. Note that I've pasted the part into a slightly different vector. The map of pBca9145-Bca1144#5 is [[Media:JCA_pBca9145-Bca1144.seq \| here]]:
-	Wobble ca9939/ca9940 (107bp, EcoRI/BamHI)	+	Wobble ca9939/ca9940 (107bp, wobpdt)
-	~~Sub into~~ pBca9145-Bca1144#5 (EcoRI/BamHI, 2057+910, L)	+	Digest wobpdt (EcoRI/BamHI, L, wobdig)
-	~~Product is~~ pBca9145-Bca9939 ~~{Ala2}~~	+	Digest pBca9145-Bca1144#5 (EcoRI/BamHI, 2057+910, L, vectdig)
		+	Ligate wobdig + vectdig (pBca9145-Bca9939)
	----		----
-	ca9939 Forward construction of Ala2 basic part	+	>ca9939 Forward construction of Ala2 basic part
	CCATAgaattcATGagatctGGGGCTATAGCTCAGCTGGGAGAGCGCCTGCTTCTAACGCAG		CCATAgaattcATGagatctGGGGCTATAGCTCAGCTGGGAGAGCGCCTGCTTCTAACGCAG
-	ca9940 Reverse construction of Ala2 basic part	+	>ca9940 Reverse construction of Ala2 basic part
	CTGATggatccTGGTGGAGCTATGCGGGATCGAACCGCAGACCTCCTGCGTTAGAAGCAGGCGCTC		CTGATggatccTGGTGGAGCTATGCGGGATCGAACCGCAGACCTCCTGCGTTAGAAGCAGGCGCTC

JCAnderson at 02:57, 25 February 2011

2011-02-25T02:57:19Z

←Older revision		Revision as of 02:57, 25 February 2011
Line 4:		Line 4:

	===Gene Synthesis===		===Gene Synthesis===
-	Sometimes you don't have the source DNA. Perhaps the sequence comes from a source organism that is not available to you. Perhaps it is a eukaryotic CDS and you wish to express it in a bacteria. In that case, you might want to change the codon usage and eliminate all the intron sequences. If you have a sequence that is over 120bp or so, and you can't simply amplify the sequence from some source DNA, your only option is Gene Synthesis. There are a number of commercial suppliers who will synthesize your part and send you plasmid DNA (but it's pretty expensive). You can also do gene synthesis on your own. There is a website called GeneDesign http://genedesign.thruhere.net/~~gdo~~/~~index~~.~~html~~ that will help you design your part and determine the oligos you should order to assemble your part.	+	Sometimes you don't have the source DNA. Perhaps the sequence comes from a source organism that is not available to you. Perhaps it is a eukaryotic CDS and you wish to express it in a bacteria. In that case, you might want to change the codon usage and eliminate all the intron sequences. If you have a sequence that is over 120bp or so, and you can't simply amplify the sequence from some source DNA, your only option is Gene Synthesis. There are a number of commercial suppliers who will synthesize your part and send you plasmid DNA (but it's pretty expensive). You can also do gene synthesis on your own. There is a website called GeneDesign http://genedesign.thruhere.net/cgi-bin/gd/gdRevTrans.cg that will help you design your part and determine the oligos you should order to assemble your part.

	===Overlap Extension===		===Overlap Extension===

JCAnderson: /* Short Part Quiz */

2011-01-27T18:37:01Z

Short Part Quiz

←Older revision		Revision as of 18:37, 27 January 2011
Line 63:		Line 63:

	==Short Part Quiz==		==Short Part Quiz==
-	Design oligos and write up 4 construction files for the following BglBricks parts. Make your parts in plasmid pBca9145-Bca1144#5 by the appropriate method. If you are given amino acid sequence rather than DNA, you'll need to design a DNA sequence for the peptide using GeneDesign (http://~~baderlab~~.~~bme~~.~~jhu.edu~~/gd/). Put all 4 construction files into a single email message one after the other in submitting your answer.	+	Design oligos and write up 4 construction files for the following BglBricks parts. Make your parts in plasmid pBca9145-Bca1144#5 by the appropriate method. If you are given amino acid sequence rather than DNA, you'll need to design a DNA sequence for the peptide using GeneDesign (http://genedesign.thruhere.net/gdo/index.html). Put all 4 construction files into a single email message one after the other in submitting your answer.
	<pre>		<pre>
	Short Description Sequence		Short Description Sequence

JCAnderson: /* Gene Synthesis */

2011-01-27T18:35:33Z

Gene Synthesis

←Older revision		Revision as of 18:35, 27 January 2011
Line 4:		Line 4:

	===Gene Synthesis===		===Gene Synthesis===
-	Sometimes you don't have the source DNA. Perhaps the sequence comes from a source organism that is not available to you. Perhaps it is a eukaryotic CDS and you wish to express it in a bacteria. In that case, you might want to change the codon usage and eliminate all the intron sequences. If you have a sequence that is over 120bp or so, and you can't simply amplify the sequence from some source DNA, your only option is Gene Synthesis. There are a number of commercial suppliers who will synthesize your part and send you plasmid DNA (but it's pretty expensive). You can also do gene synthesis on your own. There is a website called GeneDesign http://genedesign.thruhere.net/gd/ that will help you design your part and determine the oligos you should order to assemble your part.	+	Sometimes you don't have the source DNA. Perhaps the sequence comes from a source organism that is not available to you. Perhaps it is a eukaryotic CDS and you wish to express it in a bacteria. In that case, you might want to change the codon usage and eliminate all the intron sequences. If you have a sequence that is over 120bp or so, and you can't simply amplify the sequence from some source DNA, your only option is Gene Synthesis. There are a number of commercial suppliers who will synthesize your part and send you plasmid DNA (but it's pretty expensive). You can also do gene synthesis on your own. There is a website called GeneDesign http://genedesign.thruhere.net/gdo/index.html that will help you design your part and determine the oligos you should order to assemble your part.

	===Overlap Extension===		===Overlap Extension===

JCAnderson: /* Gene Synthesis */

2011-01-27T18:26:06Z

Gene Synthesis

←Older revision		Revision as of 18:26, 27 January 2011
Line 4:		Line 4:

	===Gene Synthesis===		===Gene Synthesis===
-	Sometimes you don't have the source DNA. Perhaps the sequence comes from a source organism that is not available to you. Perhaps it is a eukaryotic CDS and you wish to express it in a bacteria. In that case, you might want to change the codon usage and eliminate all the intron sequences. If you have a sequence that is over 120bp or so, and you can't simply amplify the sequence from some source DNA, your only option is Gene Synthesis. There are a number of commercial suppliers who will synthesize your part and send you plasmid DNA (but it's pretty expensive). You can also do gene synthesis on your own. There is a website called GeneDesign http://~~baderlab~~.~~bme~~.~~jhu.edu~~/gd/ that will help you design your part and determine the oligos you should order to assemble your part.	+	Sometimes you don't have the source DNA. Perhaps the sequence comes from a source organism that is not available to you. Perhaps it is a eukaryotic CDS and you wish to express it in a bacteria. In that case, you might want to change the codon usage and eliminate all the intron sequences. If you have a sequence that is over 120bp or so, and you can't simply amplify the sequence from some source DNA, your only option is Gene Synthesis. There are a number of commercial suppliers who will synthesize your part and send you plasmid DNA (but it's pretty expensive). You can also do gene synthesis on your own. There is a website called GeneDesign http://genedesign.thruhere.net/gd/ that will help you design your part and determine the oligos you should order to assemble your part.

	===Overlap Extension===		===Overlap Extension===

JCAnderson: /* Enzymatic Inverse PCR (EIPCR) */

2009-03-06T15:03:08Z

Enzymatic Inverse PCR (EIPCR)

←Older revision		Revision as of 15:03, 6 March 2009
Line 32:		Line 32:

	==Enzymatic Inverse PCR (EIPCR)==		==Enzymatic Inverse PCR (EIPCR)==
-	If your part is under than 30bp or so, your best option for constructing the part is EIPCR. In EIPCR, you are amplifying the backbone of the plasmid DNA template, pinning the part into the 5' end of your oligo, and then re-circularizing the plasmid with a single restriction site (in our case, BglII).	+	If your part is under than 30bp or so, your best option for constructing the part is EIPCR (Enzymatic Inverse PCR). In EIPCR, you are amplifying the backbone of the plasmid DNA template, pinning the part into the 5' end of your oligo, and then re-circularizing the plasmid with a single restriction site (in our case, BglII). To think about this, start by brining up pBca9145-Bca1144#5 in ApE. Change the origin to somewhere in the middle of RFP (the origin means the first base in the file, you're just spinning the circle around here) so that the RFP CDS is now split into two sections. You can now use this to predict your PCR product from EIPCR the same way you always predict PCR products. The origin of replication and amp gene will be within your final PCR product. After predicting this, you'll see there are BglII sites and 5' tails on both ends. So, if you cut with BglII the plasmid could be ligated to itself to re-form the circle. Let's give it a try:

-	~~As an example, let~~'s design a part encoding 20 A's:	+	Let's design a part encoding 20 A's:
	agatctAAAAAAAAAAAAAAAAAAAAggatcc		agatctAAAAAAAAAAAAAAAAAAAAggatcc
	Let's put it into pBca9145, so start by bring up the sequence of pBca9145-Bca1144#5 in ApE. In Ape, replace the BglII/BamHI region with the new part sequence above. You now have a map of your final product. Locate a good 20bp sequence downstream of your part. In this case I've chosen:		Let's put it into pBca9145, so start by bring up the sequence of pBca9145-Bca1144#5 in ApE. In Ape, replace the BglII/BamHI region with the new part sequence above. You now have a map of your final product. Locate a good 20bp sequence downstream of your part. In this case I've chosen:

Dirk Van Swaay: /* Short Part Quiz */

2009-01-28T20:17:17Z

Short Part Quiz

←Older revision		Revision as of 20:17, 28 January 2009
Line 63:		Line 63:

	==Short Part Quiz==		==Short Part Quiz==
-	Design oligos and write up 4 construction files for the following BglBricks parts. Make your parts in plasmid pBca9145-Bca1144#5 by the appropriate method. If you are given amino acid sequence rather than DNA, you'll need to design a DNA sequence for the peptide using GeneDesign (http://baderlab.bme.jhu.edu/gd/). Put all 3 construction files into a single email message one after the other in submitting your answer.	+	Design oligos and write up 4 construction files for the following BglBricks parts. Make your parts in plasmid pBca9145-Bca1144#5 by the appropriate method. If you are given amino acid sequence rather than DNA, you'll need to design a DNA sequence for the peptide using GeneDesign (http://baderlab.bme.jhu.edu/gd/). Put all 4 construction files into a single email message one after the other in submitting your answer.
	<pre>		<pre>
	Short Description Sequence		Short Description Sequence

JCAnderson: /* Short Part Quiz */

2009-01-28T19:10:24Z

Short Part Quiz

←Older revision		Revision as of 19:10, 28 January 2009
Line 63:		Line 63:

	==Short Part Quiz==		==Short Part Quiz==
-	Design oligos and write up 3 construction files for the following BglBricks parts. Make your parts in plasmid pBca9145 by the appropriate method. If you are given amino acid sequence rather than DNA, you'll need to design a DNA sequence for the peptide using GeneDesign (http://baderlab.bme.jhu.edu/gd/). Put all 3 construction files into a single email message one after the ~~after~~ in submitting your answer.	+	Design oligos and write up 4 construction files for the following BglBricks parts. Make your parts in plasmid pBca9145-Bca1144#5 by the appropriate method. If you are given amino acid sequence rather than DNA, you'll need to design a DNA sequence for the peptide using GeneDesign (http://baderlab.bme.jhu.edu/gd/). Put all 3 construction files into a single email message one after the other in submitting your answer.
	<pre>		<pre>
	Short Description Sequence		Short Description Sequence