Talk:20.109(S13):Context-setting and primer design (Day1) - Revision history

AgiStachowiak: /* Sensitivity */

AgiStachowiak — Tue, 19 Feb 2013 16:50:35 GMT

Sensitivity

←Older revision		Revision as of 16:50, 19 February 2013
Line 49:		Line 49:
	\|-		\|-
	\|Yellow		\|Yellow
-	\|CTGACGTGGATGCTATTC<br>Agi: ctg acg t'''a'''g atg cta '''g'''tc<br>new G/C = 50%, new Tm = 56.4	+	\|CTGACGTGGATGCTATTC<br>Agi: '''c''' ctg acg t'''a'''g atg cta '''g'''tc<br>new G/C = 50%, new Tm = 56.4 - oops, 55.1<br>added one more "c" at 5' for Tm = 57.7
	\|CCGTCAACCGTCACTGTC<br>Agi: fine		\|CCGTCAACCGTCACTGTC<br>Agi: fine
	\|bp 18		\|bp 18

AgiStachowiak: /* Specificity */

AgiStachowiak — Tue, 19 Feb 2013 16:21:38 GMT

Specificity

←Older revision		Revision as of 16:21, 19 February 2013
Line 168:		Line 168:
	\|-		\|-
	\|Green		\|Green
-	\|AATCAGGGACGAATAGCTC<br>Agi: fine	+	\|AATCAGGGACGAATAGCTC<br>Agi: fine; emailed change: GCAATCAGGGACGAATAGCTCAG
-	\|TGATAGTCCTGCGTCTTATTC<br>Agi: fine	+	\|TGATAGTCCTGCGTCTTATTC<br>Agi: fine; emailed change: AACTGATAGTCCTGCGTCTTATTCTG
	\|bp 64		\|bp 64
	\|bp 169		\|bp 169

AgiStachowiak: /* Update for everyone */

AgiStachowiak — Sat, 16 Feb 2013 17:55:01 GMT

Update for everyone

←Older revision		Revision as of 17:55, 16 February 2013
Line 3:		Line 3:
	As explained in pre-lab lecture, we were remiss (mainly me, Agi!) in not providing you with the best sequence information for ''V corneae''. Moreover, we told you to shoot for a Tm of 58 °C instead of a Tm of 63 °C (Ta of 58 °C).		As explained in pre-lab lecture, we were remiss (mainly me, Agi!) in not providing you with the best sequence information for ''V corneae''. Moreover, we told you to shoot for a Tm of 58 °C instead of a Tm of 63 °C (Ta of 58 °C).

-	Below, we have listed modified primer sequences (just under your originals) that match the Broad sequence. An ApE file of this sequence is linked [[Media:Vcorneae_SSU-Broad-direct.gb \| here]]. For comparison, the complete sequences from [[Media:Vcorneae_SUU-complete-Baker.gb \| Baker et al]] and [[Media:Vcorneae_SSU-complete-DaSilva.gb‎ \| DaSilva et al.]] are each linked. You can ''Align Sequences'' (under ''Tools'') in ApE to see the differences for yourself.~~<font color=red>In progress for WF.</font color>~~	+	Below, we have listed modified primer sequences (just under your originals) that match the Broad sequence. An ApE file of this sequence is linked [[Media:Vcorneae_SSU-Broad-direct.gb \| here]]. For comparison, the complete sequences from [[Media:Vcorneae_SUU-complete-Baker.gb \| Baker et al]] and [[Media:Vcorneae_SSU-complete-DaSilva.gb‎ \| DaSilva et al.]] are each linked. You can ''Align Sequences'' (under ''Tools'') in ApE to see the differences for yourself.

	As a default, we will order these primers on Tuesday for arrival on Thursday, and run two separate PCRs: one at Ta = 58 °C and one at Ta = 53 °C.		As a default, we will order these primers on Tuesday for arrival on Thursday, and run two separate PCRs: one at Ta = 58 °C and one at Ta = 53 °C.

AgiStachowiak: /* Sensitivity */

AgiStachowiak — Sat, 16 Feb 2013 17:54:25 GMT

Sensitivity

←Older revision		Revision as of 17:54, 16 February 2013
Line 36:		Line 36:
	\|Green		\|Green
	\|AGG TTG ATT CTG CCT GAC<br>Agi: fine		\|AGG TTG ATT CTG CCT GAC<br>Agi: fine
-	\|CGG AAC CAA ACC CTG AT<br>Agi: CGG '''T'''AC '''A'''AA ACC CT'''A''' AT<br>new G/C = 41%, new Tm = 52.8<br>Tm ~~too low, I will add a couple bases later~~	+	\|CGG AAC CAA ACC CTG AT<br>Agi: '''C''' CGG '''T'''AC '''A'''AA ACC CT'''A''' AT<br>new G/C = 44.4%, new Tm = 55.8<br>added 1 extra base to increase Tm
	\|bp 5		\|bp 5
	\|bp 236		\|bp 236

AgiStachowiak: /* Specificity */

AgiStachowiak — Sat, 16 Feb 2013 17:51:22 GMT

Specificity

←Older revision		Revision as of 17:51, 16 February 2013
Line 176:		Line 176:
	\|Orange		\|Orange
	\|CTGCCTGACGTAGATGCTAG<br>Agi: fine		\|CTGCCTGACGTAGATGCTAG<br>Agi: fine
-	\|CTGTCTTGGTAGTCCACTAC <br>Agi: CTG TCT TGG TAG TCC '''ATTAC''' AC TAC (5 insertions -- but ''no'' sequence that I found matches yours, most only require 1 insertion -- so check your work please)	+	\|CTGTCTTGGTAGTCCACTAC <br>Agi: CTG TCT TGG TAG TCC '''ATTAC''' AC TAC (5 insertions -- but ''no'' sequence that I found matches yours, most only require 1 insertion -- so check your work please)<br>new G/C = 44%, new Tm = 61.7
	\|bp 14		\|bp 14
	\|bp 203		\|bp 203

AgiStachowiak: /* Sensitivity */

AgiStachowiak — Sat, 16 Feb 2013 17:50:21 GMT

Sensitivity

←Older revision		Revision as of 17:50, 16 February 2013
Line 36:		Line 36:
	\|Green		\|Green
	\|AGG TTG ATT CTG CCT GAC<br>Agi: fine		\|AGG TTG ATT CTG CCT GAC<br>Agi: fine
-	\|CGG AAC CAA ACC CTG AT<br>Agi: CGG '''T'''AC '''A'''AA ACC CT'''A''' AT<br>new G/C = 41%, new Tm = 52.98<br>Tm too low, I will add a couple bases later	+	\|CGG AAC CAA ACC CTG AT<br>Agi: CGG '''T'''AC '''A'''AA ACC CT'''A''' AT<br>new G/C = 41%, new Tm = 52.8<br>Tm too low, I will add a couple bases later
	\|bp 5		\|bp 5
	\|bp 236		\|bp 236

AgiStachowiak: /* Sensitivity */

AgiStachowiak — Sat, 16 Feb 2013 17:49:52 GMT

Sensitivity

←Older revision		Revision as of 17:49, 16 February 2013
Line 135:		Line 135:
	\|Red		\|Red
	\|5' CCA GGT TGA TTC TGC CTG 3' <br>Agi:fine		\|5' CCA GGT TGA TTC TGC CTG 3' <br>Agi:fine
-	\|5' CTC TCC GGA ACC AAA CC 3' <br>Agi: CTC TCC GG'''T''' AC'''A''' AAA CC	+	\|5' CTC TCC GGA ACC AAA CC 3' <br>Agi: CTC TCC GG'''T''' AC'''A''' AAA CC<br>new G/C = 53%, new Tm = 55.6
	\|bp 9, F		\|bp 9, F
	\|bp 225, R		\|bp 225, R

AgiStachowiak: /* Sensitivity */

AgiStachowiak — Sat, 16 Feb 2013 17:48:33 GMT

Sensitivity

←Older revision		Revision as of 17:48, 16 February 2013
Line 120:		Line 120:
	\|-		\|-
	\|Purple		\|Purple
-	\|5' GGACGGCTCAGTGATAG 3'	+	\|5' GGACGGCTCAGTGATAG 3'<br>Agi:fine for EH
-	\|5' CTACCACACCCAGAACTTAC 3'	+	\|5' CTACCACACCCAGAACTTAC 3'<br>Agi:fine for EH
	\|bp 84, F		\|bp 84, F
	\|bp 167, R		\|bp 167, R
Line 127:		Line 127:
	\|-		\|-
	\|Yellow		\|Yellow
-	\|5' GAGAAGTAAGATGTTTAGCAAGTAT 3'	+	\|5' GAGAAGTAAGATGTTTAGCAAGTAT 3'<br>Agi:fine for EH
-	\|5' CATCACCACAAAAGTCCAA 3'	+	\|5' CATCACCACAAAAGTCCAA 3'<br>Agi:fine for EH
	\|bp 359, F		\|bp 359, F
	\|bp 630, R		\|bp 630, R
Line 141:		Line 141:
	\|-		\|-
	\|Platinum		\|Platinum
-	\| 5' CAT GTT GAT TCT GCC TGA C 3'	+	\| 5' CAT GTT GAT TCT GCC TGA C 3'<br>Agi:fine for EH
-	\| 5' CTC TCC TGA ACC AAA CCC TG 3'	+	\| 5' CTC TCC TGA ACC AAA CCC TG 3'<br>Agi:fine for EH
	\| bp 4, F		\| bp 4, F
	\| bp 278, R		\| bp 278, R

AgiStachowiak: /* Specificity */

AgiStachowiak — Fri, 15 Feb 2013 22:14:34 GMT

Specificity

←Older revision		Revision as of 22:14, 15 February 2013
Line 92:		Line 92:
	\|Blue		\|Blue
	\|CCGCAATCAGGGACGAA<br>Agi: fine		\|CCGCAATCAGGGACGAA<br>Agi: fine
-	\|GTCAACGTCACTGTCTTGG<br>Agi: GTC AAC '''C''' GTC ACT GTC TTG G (insertion) <br>new G/C = 55%, new Tm = 64.3	+	\|GTCAACGTCACTGTCTTGG<br>Agi: GTC AAC '''C''' GTC ACT GTC TTG G (insertion) <br>new G/C = 55%, new Tm = 61.8
	\|60		\|60
	\|195		\|195

AgiStachowiak: /* Specificity */

AgiStachowiak — Fri, 15 Feb 2013 22:13:48 GMT

Specificity

←Older revision		Revision as of 22:13, 15 February 2013
Line 78:		Line 78:
	\|Red		\|Red
	\|GGT TGA TTC TGC CTG ACG TGG<br>Agi:fine		\|GGT TGA TTC TGC CTG ACG TGG<br>Agi:fine
-	\|CCT TCG TCC TTC ATC ACC ACA A<br>Agi: CCT TCG TCC TT'''G''' ATC ACC '''GA'''A '''T''' (yours doesn't match ''any'' known sequence in last 4 bp -- do I have right region?)	+	\|CCT TCG TCC TTC ATC ACC ACA A<br>Agi: CCT TCG TCC TT'''G''' ATC ACC '''GA'''A '''T''' (yours doesn't match ''any'' known sequence in last 4 bp -- do I have right region?)<br>new G/C = 50%, new Tm = 63.5
	\|bp 6, F		\|bp 6, F
	\|bp 588, R		\|bp 588, R