User:Nkuldell/VitaYeast - Revision history

Nkuldell: /* Initial communication about strains with Leslie Mitchell in Jef's lab */

Nkuldell — Sun, 06 Jan 2013 14:23:53 GMT

Initial communication about strains with Leslie Mitchell in Jef's lab

←Older revision		Revision as of 14:23, 6 January 2013
Line 31:		Line 31:
	Leslie		Leslie
	[[Image:Beta carotene strains.jpg\|300px]]<br>		[[Image:Beta carotene strains.jpg\|300px]]<br>
-	Note: CEN.PK strain is described [http://wiki.yeastgenome.org/index.php/Commonly_used_strains#CEN.PK_.28aka_CEN.PK2.29 here]<br>[[Image:CEN.PK2 genotype.png\|600px]]	+	Note: CEN.PK strain is described [http://wiki.yeastgenome.org/index.php/Commonly_used_strains#CEN.PK_.28aka_CEN.PK2.29 here]<br>[[Image:CEN.PK2 genotype.png\|600px]]<br>
		+	NOTE: there seems to be a mix up between 128 and 179 from the picture above and what I streaked out in the MIT lab. Strain "128" is the one that's more orange than yellow and that gives off white, yellow and red colonies and that's trp-. This strain was stored in my lab collection as <b>NY458. </b>

	==Getting started==		==Getting started==

Nkuldell: /* BTS1 */

Nkuldell — Wed, 29 Aug 2012 18:58:09 GMT

BTS1

←Older revision		Revision as of 18:58, 29 August 2012
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	*BTS1-ORF-FWD		*BTS1-ORF-FWD
	**ATGGAGGCCA AGATAGATGA GCTGATCAAT AATGATCCTG		**ATGGAGGCCA AGATAGATGA GCTGATCAAT AATGATCCTG
		+	**NO310
	*BTS1-ORF-REV		*BTS1-ORF-REV
	**5'- TCA CAA TTC GGA TAA GTG GTC TAT TAT ATA TAA CAA TTC -3'		**5'- TCA CAA TTC GGA TAA GTG GTC TAT TAT ATA TAA CAA TTC -3'
		+	**NO311

Nkuldell at 19:08, 22 August 2012

Nkuldell — Wed, 22 Aug 2012 19:08:40 GMT

←Older revision		Revision as of 19:08, 22 August 2012
Line 88:		Line 88:
	**TCA GAA AGC AAG AAC ACC AAC GGA TCG AGC GAT CAC GGC CCC AAT ((made longer than in paper to improve confidence in PCR amplification from dirty template)		**TCA GAA AGC AAG AAC ACC AAC GGA TCG AGC GAT CAC GGC CCC AAT ((made longer than in paper to improve confidence in PCR amplification from dirty template)
	**NO309		**NO309
		+	====BTS1====
		+	*SGD entry [http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=BTS1 here]
		+	*Gene/seq resources from SGD [http://www.yeastgenome.org/cgi-bin/seqTools?seqname=YPL069C here]
		+	*BTS1-ORF-FWD
		+	**ATGGAGGCCA AGATAGATGA GCTGATCAAT AATGATCCTG
		+	*BTS1-ORF-REV
		+	**5'- TCA CAA TTC GGA TAA GTG GTC TAT TAT ATA TAA CAA TTC -3'

Nkuldell: /* CrtI */

Nkuldell — Tue, 21 Aug 2012 16:55:31 GMT

CrtI

←Older revision		Revision as of 16:55, 21 August 2012
Line 84:		Line 84:
	*crtI-ORF-F (made longer than in paper to improve confidence in PCR amplification from dirty template)		*crtI-ORF-F (made longer than in paper to improve confidence in PCR amplification from dirty template)
	**atgggaaaagaacaagatcaggataaacccacagctatcat		**atgggaaaagaacaagatcaggataaacccacagctatcat
		+	**NO308
	*crtI-ORF-R		*crtI-ORF-R
	**TCA GAA AGC AAG AAC ACC AAC GGA TCG AGC GAT CAC GGC CCC AAT ((made longer than in paper to improve confidence in PCR amplification from dirty template)		**TCA GAA AGC AAG AAC ACC AAC GGA TCG AGC GAT CAC GGC CCC AAT ((made longer than in paper to improve confidence in PCR amplification from dirty template)
		+	**NO309

Nkuldell: /* CrtE */

Nkuldell — Tue, 21 Aug 2012 16:55:05 GMT

CrtE

←Older revision		Revision as of 16:55, 21 August 2012
Line 68:		Line 68:
	*crtE-ORF-F		*crtE-ORF-F
	**atggattacgcgaacatcctcacagcaattccactcgagtttac (made longer than published oligo to help with PCR amplification from dirty template)		**atggattacgcgaacatcctcacagcaattccactcgagtttac (made longer than published oligo to help with PCR amplification from dirty template)
		+	**NO306
	*crtE-ORF-R		*crtE-ORF-R
-
	** TCA CAG AGG GAT ATC GGC TAG CTT TTT CAG GAT CGC TTC CAA TTT GAC G (made longer than published oligo to help with PCR amplification from dirty template)		** TCA CAG AGG GAT ATC GGC TAG CTT TTT CAG GAT CGC TTC CAA TTT GAC G (made longer than published oligo to help with PCR amplification from dirty template)
		+	**NO307

	====CrtI====		====CrtI====

Nkuldell: /* CrtE */

Nkuldell — Sat, 18 Aug 2012 01:42:13 GMT

CrtE

←Older revision		Revision as of 01:42, 18 August 2012
Line 67:		Line 67:
	*also part # [http://partsregistry.org/Part:BBa_K530001:Design BBa_K530001] available in NB481 (CamR)		*also part # [http://partsregistry.org/Part:BBa_K530001:Design BBa_K530001] available in NB481 (CamR)
	*crtE-ORF-F		*crtE-ORF-F
-	**~~CGCGGATCCATGGATTACGCGAACATCCTC~~	+	**atggattacgcgaacatcctcacagcaattccactcgagtttac (made longer than published oligo to help with PCR amplification from dirty template)
	*crtE-ORF-R		*crtE-ORF-R
-	**~~TGCGGTCGACTCACAGAGGGATATCGGCTAG~~	+
-	*crtE-SEQ-	+	** TCA CAG AGG GAT ATC GGC TAG CTT TTT CAG GAT CGC TTC CAA TTT GAC G (made longer than published oligo to help with PCR amplification from dirty template)
		+
	====CrtI====		====CrtI====
	*Genomic CrtI seq from X. dendrorhous is [http://www.ncbi.nlm.nih.gov/nuccore/AY177424.1 here]		*Genomic CrtI seq from X. dendrorhous is [http://www.ncbi.nlm.nih.gov/nuccore/AY177424.1 here]

Nkuldell: /* CrtI */

Nkuldell — Sat, 18 Aug 2012 01:34:43 GMT

CrtI

←Older revision		Revision as of 01:34, 18 August 2012
Line 80:		Line 80:
	**NO301		**NO301
	**GCCGGTACCGGCCGGCCGCAAATTAAAGCCTTCGAGCG		**GCCGGTACCGGCCGGCCGCAAATTAAAGCCTTCGAGCG
-	*crtI-ORF-F	+	*crtI-ORF-F (made longer than in paper to improve confidence in PCR amplification from dirty template)
-	**~~GCGGGATCCATGGGAAAAGAACAAGATCAGG~~	+	**atgggaaaagaacaagatcaggataaacccacagctatcat
	*crtI-ORF-R		*crtI-ORF-R
-	**~~TGCGGTCGACTCAGAAAGCAAGAACACCAACG~~	+	**TCA GAA AGC AAG AAC ACC AAC GGA TCG AGC GAT CAC GGC CCC AAT ((made longer than in paper to improve confidence in PCR amplification from dirty template)
-	*crtI-SEQ	+

Nkuldell at 01:18, 18 August 2012

Nkuldell — Sat, 18 Aug 2012 01:18:03 GMT

←Older revision		Revision as of 01:18, 18 August 2012
Line 50:		Line 50:
	====CrtYB====		====CrtYB====
	*Genomic CrtYB seq from X. dendrorhous is [http://www.ncbi.nlm.nih.gov.libproxy.mit.edu/nuccore/AY177204.1 here]		*Genomic CrtYB seq from X. dendrorhous is [http://www.ncbi.nlm.nih.gov.libproxy.mit.edu/nuccore/AY177204.1 here]
-	*also part # [http://partsregistry.org/Part:BBa_K530000:Design BBa_K530000] available?	+	*also part # [http://partsregistry.org/Part:BBa_K530000:Design BBa_K530000] available in NB480 (CamR)
	*Pr-crtYB-Tr-F		*Pr-crtYB-Tr-F
	**NO298		**NO298
Line 63:		Line 63:
	**NO303		**NO303
	**5'- TTA CTG CCC TTC CCA TCC GCT CAT GAC CAC ACT CAA GAC TTT CCG TAC -3'		**5'- TTA CTG CCC TTC CCA TCC GCT CAT GAC CAC ACT CAA GAC TTT CCG TAC -3'
		+	====CrtE====
		+	*Genomic CrtE seq from X. dendrorhous is [http://www.ncbi.nlm.nih.gov/nuccore/DQ016502.1 here]
		+	*also part # [http://partsregistry.org/Part:BBa_K530001:Design BBa_K530001] available in NB481 (CamR)
		+	*crtE-ORF-F
		+	**CGCGGATCCATGGATTACGCGAACATCCTC
		+	*crtE-ORF-R
		+	**TGCGGTCGACTCACAGAGGGATATCGGCTAG
		+	*crtE-SEQ-
	====CrtI====		====CrtI====
		+	*Genomic CrtI seq from X. dendrorhous is [http://www.ncbi.nlm.nih.gov/nuccore/AY177424.1 here]
		+	*also part # [http://partsregistry.org/Part:BBa_K530002:Design BBa_K530002] available in NB482 (CamR)
	*Pr-crtI-Tr-F		*Pr-crtI-Tr-F
	**NO300		**NO300
Line 70:		Line 80:
	**NO301		**NO301
	**GCCGGTACCGGCCGGCCGCAAATTAAAGCCTTCGAGCG		**GCCGGTACCGGCCGGCCGCAAATTAAAGCCTTCGAGCG
-	~~===Yeast Colony PCR===~~	+	*crtI-ORF-F
-	~~From 20.109(F08) expt....~~	+	**GCGGGATCCATGGGAAAAGAACAAGATCAGG
-	~~#Use the microwave to release the DNA from the yeast. On the tip of a sterile toothpick, pick~~-~~up 1/2 of the colony that is candidate and swirl the cells in 20 μl of sterile water in an eppendorf tube.~~	+	*crtI-ORF-R
-	~~#Close the cap and microwave tube in an eppendorf rack for 15 seconds.~~	+	**TGCGGTCGACTCAGAAAGCAAGAACACCAACG
-	~~#Move 2.5 μl of the microwaved mixes, yeast debris and all, into a 200 μl PCR tube.~~	+	*crtI-SEQ
-	~~#Prepare PCR cocktails. Each reaction will include:~~	+
-	#*~~10 μl 2.5X PCR mastermix~~	+
-	#*~~0.5 μl forward primer NO298 or NO300~~	+
-	#*~~0.5 μl reverse primer NO299 or NO301~~	+
-	#*~~water to a final volume of 22.5 μl.~~	+
-	~~#Add 22.5 μl of PCR cocktail to each PCR tube with yeast.~~	+
-	~~#Cycle the reactions as:~~	+
-	#*~~95° 4 minutes~~	+
-	#*~~95° 1 minute~~	+
-	~~#*52° 1 minute~~	+
-	~~#*72° 4 minutes~~	+
-	~~#*repeat steps 2~~-~~4 35 times~~	+
-	~~#*72° 10 minutes~~	+
-	~~#*4° forever~~	+

Nkuldell: /* Summer 2012 Experiments */

Nkuldell — Wed, 27 Jun 2012 14:35:07 GMT

Summer 2012 Experiments

←Older revision		Revision as of 14:35, 27 June 2012
Line 57:		Line 57:
	**NO299		**NO299
	**TTTTCTGCAGGCGGCCGCGCAAATTAAAGCCTTCGAGCG		**TTTTCTGCAGGCGGCCGCGCAAATTAAAGCCTTCGAGCG
		+	*crtYB-ORF-F
		+	**NO302
		+	**5'- ATG ACG GCT CTC GCA TAT TAC CAG ATC CAT CTG ATC TAT ACT CTC CC -3'
		+	*crtYB-ORF-R
		+	**NO303
		+	**5'- TTA CTG CCC TTC CCA TCC GCT CAT GAC CAC ACT CAA GAC TTT CCG TAC -3'
		+	====CrtI====
	*Pr-crtI-Tr-F		*Pr-crtI-Tr-F
	**NO300		**NO300

Nkuldell: /* Summer 2012 Experiments */

Nkuldell — Tue, 26 Jun 2012 11:17:10 GMT

Summer 2012 Experiments

←Older revision		Revision as of 11:17, 26 June 2012
Line 48:		Line 48:
	==Summer 2012 Experiments==		==Summer 2012 Experiments==
	===Primers===		===Primers===
		+	====CrtYB====
		+	*Genomic CrtYB seq from X. dendrorhous is [http://www.ncbi.nlm.nih.gov.libproxy.mit.edu/nuccore/AY177204.1 here]
		+	*also part # [http://partsregistry.org/Part:BBa_K530000:Design BBa_K530000] available?
	*Pr-crtYB-Tr-F		*Pr-crtYB-Tr-F
	**NO298		**NO298