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

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==T/R section==
+
==Update for everyone==
 +
 
 +
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.
 +
 
 +
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.
 +
 
 +
However, if you would like to revise your primers to a Tm of 63 °C, we will accept these new designs through Monday at 9 pm.
 +
 
 +
==T/R section (primer check in progress)==
Please sign up for one of the design challenges -- four teams per challenge -- right away.
Please sign up for one of the design challenges -- four teams per challenge -- right away.
Line 18: Line 28:
|-
|-
| Grey (Platinum) :)  
| Grey (Platinum) :)  
-
| CAT GCT TGC CAA CAC AG
+
| CAT GCT TGC CAA CAC AG<br>Agi: PT so unchanged
-
| GAA TGG TCT GGT TGC TTC  
+
| GAA TGG TCT GGT TGC TTC<br>Agi: PT so unchanged
| bp 9
| bp 9
| bp 251
| bp 251
Line 25: Line 35:
|-
|-
|Green
|Green
-
|AGG TTG ATT CTG CCT GAC  
+
|AGG TTG ATT CTG CCT GAC<br>Agi: fine
-
|CGG AAC CAA ACC CTG AT
+
|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
Line 32: Line 42:
|-
|-
|Pink
|Pink
-
|ttctgcctgacgtagatg
+
|ttctgcctgacgtagatg<br>Agi: fine
-
|cctctccggaaccaaac
+
|cctctccggaaccaaac<br>Agi: CCT CTC CGG '''T'''AC '''A'''AA AC <br>new G/C = 52.9%, new Tm = 55.6
|bp 12
|bp 12
|bp 226
|bp 226
Line 39: Line 49:
|-
|-
|Yellow
|Yellow
-
|CTGACGTGGATGCTATTC
+
|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
+
|CCGTCAACCGTCACTGTC<br>Agi: fine
|bp 18
|bp 18
|bp 199
|bp 199
Line 60: Line 70:
|-  
|-  
| Purple
| Purple
-
|5' GCG ATG ATT TAG TCT GGC 3'
+
|5' GCG ATG ATT TAG TCT GGC 3'<br>Agi: fine
-
|5’ CAA TTT CCA ACG ACC ATG C 3’
+
|5’ CAA TTT CCA ACG ACC ATG C 3’<br>Agi: fine
| bp 93, F
| bp 93, F
| bp 879, R
| bp 879, R
Line 67: Line 77:
|-
|-
|Red
|Red
-
|GGT TGA TTC TGC CTG ACG TGG  
+
|GGT TGA TTC TGC CTG ACG TGG<br>Agi:fine
-
|CCT TCG TCC TTC ATC ACC ACA A
+
|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
Line 74: Line 84:
|-
|-
|Orange
|Orange
-
|5'cgt agt cat aga agg gca aag ag3'
+
|5'cgt agt cat aga agg gca aag ag3'<br>Agi: fine
-
|5' ctc tct ctc tac ctg cta ttg ta 3'
+
|5' ctc tct ctc tac ctg cta ttg ta 3'<br>Agi: fine
|441
|441
|1005
|1005
Line 81: Line 91:
|-
|-
|Blue
|Blue
-
|CCGCAATCAGGGACGAA
+
|CCGCAATCAGGGACGAA<br>Agi: fine
-
|GTCAACGTCACTGTCTTGG
+
|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
Line 110: Line 120:
|-
|-
|Purple
|Purple
-
|5' GGA CGG CTC AGT GAT AG 3'
+
|5' GGACGGCTCAGTGATAG 3'<br>Agi:fine for EH
-
|
+
|5' CTACCACACCCAGAACTTAC 3'<br>Agi:fine for EH
-
|
+
|bp 84, F
-
|
+
|bp 167, R
-
|
+
|EH, SSU rRNA
|-
|-
|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 124: Line 134:
|-
|-
|Red
|Red
-
|5' CCA GGT TGA TTC TGC CTG 3'  
+
|5' CCA GGT TGA TTC TGC CTG 3' <br>Agi:fine
-
|5' CTC TCC GGA ACC AAA CC 3'
+
|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
Line 131: 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
Line 151: Line 161:
|-
|-
| Pink
| Pink
-
|TTCTGCCTGACGTAGATG  
+
|TTCTGCCTGACGTAGATG<br>Agi: fine
-
|TCAACCGTCACTGTCTTG  
+
|TCAACCGTCACTGTCTTG<br>Agi: fine
|bp 12
|bp 12
|bp 213
|bp 213
Line 158: Line 168:
|-
|-
|Green
|Green
-
|AATCAGGGACGAATAGCTC
+
|AATCAGGGACGAATAGCTC<br>Agi: fine; emailed change: GCAATCAGGGACGAATAGCTCAG
-
|TGATAGTCCTGCGTCTTATTC
+
|TGATAGTCCTGCGTCTTATTC<br>Agi: fine; emailed change: AACTGATAGTCCTGCGTCTTATTCTG
|bp 64
|bp 64
|bp 169
|bp 169
Line 165: Line 175:
|-
|-
|Orange
|Orange
-
|CTGCCTGACGTAGATGCTAG
+
|CTGCCTGACGTAGATGCTAG<br>Agi: fine
-
|CTGTCTTGGTAGTCCACTAC
+
|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
Line 172: Line 182:
|-
|-
| Blue
| Blue
-
| ACGAGCAATACAGGAGTGG
+
| ACGAGCAATACAGGAGTGG<br>Agi: fine
-
| TCCATCTGAGCACCTTCC
+
| TCCATCTGAGCACCTTCC<br>Agi: fine
| bp 841
| bp 841
| bp 1049
| bp 1049

Current revision

Contents

Update for everyone

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 here. For comparison, the complete sequences from Baker et al and 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.

However, if you would like to revise your primers to a Tm of 63 °C, we will accept these new designs through Monday at 9 pm.

T/R section (primer check in progress)

Please sign up for one of the design challenges -- four teams per challenge -- right away.

When you have finished, post your primer sequences (from 5' to 3') so that they can be ordered.

I will name your primers by the approximate base-pair that they target in V corneae or E hellem, so please share this information as well.

Sensitivity

Team color Forward (F) primer Reverse (R) primer Target site (e.g. bp 60), F Target side (e.g., bp 300), R Is bp # for VC or EH? Which gene?
Grey (Platinum) :) CAT GCT TGC CAA CAC AG
Agi: PT so unchanged
GAA TGG TCT GGT TGC TTC
Agi: PT so unchanged
bp 9 bp 251 E. Hellem: polar tube protein gene
Green AGG TTG ATT CTG CCT GAC
Agi: fine
CGG AAC CAA ACC CTG AT
Agi: C CGG TAC AAA ACC CTA AT
new G/C = 44.4%, new Tm = 55.8
added 1 extra base to increase Tm
bp 5 bp 236 VC: SSU rRNA
Pink ttctgcctgacgtagatg
Agi: fine
cctctccggaaccaaac
Agi: CCT CTC CGG TAC AAA AC
new G/C = 52.9%, new Tm = 55.6
bp 12 bp 226 VC
Yellow CTGACGTGGATGCTATTC
Agi: c ctg acg tag atg cta gtc
new G/C = 50%, new Tm = 56.4 - oops, 55.1
added one more "c" at 5' for Tm = 57.7
CCGTCAACCGTCACTGTC
Agi: fine
bp 18 bp 199 VC small subunit ribosomal RNA gene


Specificity

Team Color Forward primer Reverse primer Target site (e.g. bp 60), F Target side (e.g., bp 300), R Is bp # for VC or EH? Which gene?
Purple 5' GCG ATG ATT TAG TCT GGC 3'
Agi: fine
5’ CAA TTT CCA ACG ACC ATG C 3’
Agi: fine
bp 93, F bp 879, R VC, SSU rRNA
Red GGT TGA TTC TGC CTG ACG TGG
Agi:fine
CCT TCG TCC TTC ATC ACC ACA A
Agi: CCT TCG TCC TTG ATC ACC GAA T (yours doesn't match any known sequence in last 4 bp -- do I have right region?)
new G/C = 50%, new Tm = 63.5
bp 6, F bp 588, R VC, SSU RNA
Orange 5'cgt agt cat aga agg gca aag ag3'
Agi: fine
5' ctc tct ctc tac ctg cta ttg ta 3'
Agi: fine
441 1005 V. corneae TV4/TV5 SSU rRNA
Blue CCGCAATCAGGGACGAA
Agi: fine
GTCAACGTCACTGTCTTGG
Agi: GTC AAC C GTC ACT GTC TTG G (insertion)
new G/C = 55%, new Tm = 61.8
60 195 V. corneae TV4/TV5 SSU rRNA


W/F section

Please sign up for one of the design challenges -- four teams per challenge -- right away.

When you have finished, post your primer sequences (from 5' to 3') so that they can be ordered.

I will name your primers by the approximate base-pair that they target in V corneae or E hellem, so please share this information as well.

Sensitivity

Team color Forward (F) primer Reverse (R) primer Target site (e.g. bp 60), F Target side (e.g., bp 300), R Is bp # for VC or EH? Which gene?
Purple 5' GGACGGCTCAGTGATAG 3'
Agi:fine for EH
5' CTACCACACCCAGAACTTAC 3'
Agi:fine for EH
bp 84, F bp 167, R EH, SSU rRNA
Yellow 5' GAGAAGTAAGATGTTTAGCAAGTAT 3'
Agi:fine for EH
5' CATCACCACAAAAGTCCAA 3'
Agi:fine for EH
bp 359, F bp 630, R EH, SSU rRNA
Red 5' CCA GGT TGA TTC TGC CTG 3'
Agi:fine
5' CTC TCC GGA ACC AAA CC 3'
Agi: CTC TCC GGT ACA AAA CC
new G/C = 53%, new Tm = 55.6
bp 9, F bp 225, R VC, SSU, rRNA
Platinum 5' CAT GTT GAT TCT GCC TGA C 3'
Agi:fine for EH
5' CTC TCC TGA ACC AAA CCC TG 3'
Agi:fine for EH
bp 4, F bp 278, R EH, SSU rRNA


Specificity

Team Color Forward primer Reverse primer Target site (e.g. bp 60), F Target side (e.g., bp 300), R Is bp # for VC or EH? Which gene?
Pink TTCTGCCTGACGTAGATG
Agi: fine
TCAACCGTCACTGTCTTG
Agi: fine
bp 12 bp 213 for VC TV4 SSU rRNA
Green AATCAGGGACGAATAGCTC
Agi: fine; emailed change: GCAATCAGGGACGAATAGCTCAG
TGATAGTCCTGCGTCTTATTC
Agi: fine; emailed change: AACTGATAGTCCTGCGTCTTATTCTG
bp 64 bp 169 VC SSU rRNA, full sequence
Orange CTGCCTGACGTAGATGCTAG
Agi: fine
CTGTCTTGGTAGTCCACTAC
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)
new G/C = 44%, new Tm = 61.7
bp 14 bp 203 VC TV4 SSU rRNA gene
Blue ACGAGCAATACAGGAGTGG
Agi: fine
TCCATCTGAGCACCTTCC
Agi: fine
bp 841 bp 1049 VC SSU rRNA


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