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

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