Talk:20.109(S13):Context-setting and primer design (Day1): Difference between revisions
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|- | |- | ||
|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?) | ||
|bp 6, F | |bp 6, F | ||
|bp 588, R | |bp 588, R | ||
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|- | |- | ||
|Blue | |Blue | ||
|CCGCAATCAGGGACGAA | |CCGCAATCAGGGACGAA<br>Agi: fine | ||
|GTCAACGTCACTGTCTTGG | |GTCAACGTCACTGTCTTGG<bra>Agi: GTC AAC '''C''' GTC ACT GTC TTG G (insertion) | ||
|60 | |60 | ||
|195 | |195 |
Revision as of 12:20, 15 February 2013
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.in progress
As a default, we will order these primers on Tuesday and Wednesday for arrival on Thursday and Friday, 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 for T/R section and Tuesday at 9 pm for W/F section.
T/R 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? |
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: CGG TAC AAA ACC CTA AT |
bp 5 | bp 236 | VC: SSU rRNA |
Pink | ttctgcctgacgtagatg Agi: fine |
cctctccggaaccaaac Agi: CCT CTC CGG TAC AAA AC |
bp 12 | bp 226 | VC |
Yellow | CTGACGTGGATGCTATTC Agi: ctg acg tag atg cta gtc |
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?) |
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<bra>Agi: GTC AAC C GTC ACT GTC TTG G (insertion) | 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' | 5' CTACCACACCCAGAACTTAC 3' | bp 84, F | bp 167, R | EH, SSU rRNA |
Yellow | 5' GAGAAGTAAGATGTTTAGCAAGTAT 3' | 5' CATCACCACAAAAGTCCAA 3' | bp 359, F | bp 630, R | EH, SSU rRNA |
Red | 5' CCA GGT TGA TTC TGC CTG 3' | 5' CTC TCC GGA ACC AAA CC 3' | bp 9, F | bp 225, R | VC, SSU, rRNA |
Platinum | 5' CAT GTT GAT TCT GCC TGA C 3' | 5' CTC TCC TGA ACC AAA CCC TG 3' | 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 | TCAACCGTCACTGTCTTG | bp 12 | bp 213 | for VC TV4 SSU rRNA |
Green | AATCAGGGACGAATAGCTC | TGATAGTCCTGCGTCTTATTC | bp 64 | bp 169 | VC SSU rRNA, full sequence |
Orange | CTGCCTGACGTAGATGCTAG | CTGTCTTGGTAGTCCACTAC | bp 14 | bp 203 | VC TV4 SSU rRNA gene |
Blue | ACGAGCAATACAGGAGTGG | TCCATCTGAGCACCTTCC | bp 841 | bp 1049 | VC SSU rRNA |