primers


 * __Bak knockout screen:__ Three primers, in theory can be used together but better results when WT and KO are performed separately as smaller KO band can out-compete WT in heterozygotes. **
 * WT = 1.2 kb, KO = 500 bp **
 * Run: 55C x 1 min, 72C x 2 min = 35 cycles **
 * Bak geno WT: GGTGTCCACACTAGAGAACTACTC **
 * Bak geno shared: GAGCCATGAAGATGTTTAGC **
 * Bak KO R1: TCAGGACATAGCGTTGGCTAC **

WT = 304 bp, KO = 507 bp Run: 56C x 1 min, 72C x 1 min = 35 cycles Bax KO F1: GTTGACCAGAGTGGCGTAGG Bax WT F1: CCGCTTCCATTGCTCAGCGG Bax shared R1: GAGCTGATCAGAACCATCATG
 * __Bax knockout screen:__** Three primers, works best when WT and KO reactions run separately.
 * I usually anneal for 45 sec - ADD

WT = 205 bp, KO ~300 bp Run: 63C x 30 sec, 72C x 30 sec = 35 cycles Bcl-XL2F: CCG ATT GTT CAG GAG ACC TTC CTG GCT TC Bcl-XL2R2: GAA CTT GCT GCT CTC ATA GGT TTT AAG CCA AG
 * __Bcl-x floxed screen:__** Two primers yielding different sized bands for WT and floxed alleles reflecting ~100 bp insertion containing loxP site

__**Bim knockout screen:**__ Three primers can be used together or separately. WT = 400 bp, KO = 540 bp Run: 58C x 45 sec, 72C x 1 min = 35 cycles Bim geno WT: GTG CTA ACT GAA ACC AGA TTA Bim geno KO: CTC AGT CCA TTC ATC AAC AG Bim geno shared: CAT TCT CGT AAG TCC GAG TCT
 * This rxn results in a smear with some batches of homemade Taq, best to use commercial Taq to avoid variation. -ADD

~300 bp product Run: 60C x 30 sec, 72C x 1 min = 35 cycles CD4Cre F2: CD4Cre R1: CCTGGCGATCCCTGAACATGTCC
 * __CD4Cre screen:__** Two primers to detect CD4Cre.
 * Can shorten extension time to 30 sec. - ADD
 * Often see primer dimers around same size as band, but real band is obvious if reaction works well, which it usually does. -ADD

__**cFLIP floxed screen:**__ Two primers, produces separate WT and floxed bands in one reaction. cFLIP SAF + cFLIP Floxed R2 = 480 bp (floxed), 340 bp (WT) FLIPSAF1 5' CAT GAG CAC TGA GGG ACA CAG CAC 3' FLIPFloxR2 5' CGG AGT TTG CTA CAG GAA GGC CAC 3' Run: 61C – 30 sec, 72C – 1 min = 35 cycles
 * This PCR produces a lot of false negatives - the floxed (upper) band often doesn't show up if the WT (lower) band is present, so heterozygotes often appear to be homozygous WT. - CLG

Up5’-A:** GAGGTTGAGGGACTTGGCATG ** SSI-3’: ** TCAGCAGGACCCTATAATCAG **
 * __cFLIP Tg screen (both long and short):__**** Two primers, band is the same for either long or short Tg. **
 * Up5’ A + SS1 3’ = less than 1 kb **
 * Run: 52C – 30 sec, 72C – 2 min = 35 cycles **


 * __ER-Cre screen:__**** Detects ER-Cre. **
 * Run: 58C – 30 sec, 72C – 1 min = 35 cycles **
 * band is ~500kb **
 * ERCre F1: AACCTGGATAGTGAAACAGGGGC **
 * ERCre R1: GAATCTCCAGCCAGGCACACTC **

**__ER-Cre screen (new primers from Qi-Jing):__** Detects ER-Cre.

Run: 61C - 30 sec, 72C - 45 sec = 35 cycles band is 500 kb Cre-ER F: CCACCAGCCAGCTATCAACT Cre-ER R: TGAACCAGCTCCCTATCTGC

Primer stock is 1 ug/ml and working stock is 100 ng/ml (instead of 10 uM). Still use 0.5 ul of each primer for a 25 ul reaction (works out about the same).

band is < 1 kb Run: 57C x 30 sec, 72C x 45 sec = 35 cycles Lck-p: gca gga agt ggg taa cta gac taa c Mx3: tct ccc acc gtc agt acg tga gat at
 * __LckCre screen:__** Detects LckCre (Mx3 is within Cre, Lck-p is Lck-specific)

__**Lpr screen:**__ ** Three primers, should always do two separate reactions for WT and mutant because products are too close in size. ** Fas F1** : GTAAATAATTGTGCTTCGTCAG ** Fas R1** : TAGAAAGGTGCACGGGTGTG ** Fas R2** : CAAATCTAGGCATTAACAGTG **
 * Fas F1 + Fas R1 = mutant allele, 217 bp **
 * Fas F1 + Fas R2 = WT allele, 179 bp **
 * Run: 59C – 30 sec, 72C – 1 min = 35 cycles **

LTAF** : CCTTGTTGGTAAACTTCTGCC ** LTAR** : AAGAGAGCACAAGACATTGGG **
 * __LTa screen:__**** Detects WT allele of LTa. **
 * LTAF + LTAR = **
 * Run: 53C – 30 sec, 72C – 1 min = 35 cycles **

__**LysM-Cre screen:**__ ** Three primers in one reaction, produces separate WT and floxed bands. ** IMR 3066** : CCCAGAAATGCCAGATTACG ** IMR 3067** : CTTGGGCTGCCAGAATTTCTC ** IMR 3068** : TTACAGTCGGCCAGGCTGAC **
 * IMR 3066 (LysM-Cre) + IMR 3067 (Shared) + IMR 3068 (WT) = 700 bp (LysM-Cre), 350 bp (WT) **
 * Run: 63C – 1 min, 72C – 1 min = 35 cycles **


 * Began having trouble with this reaction – top band not showing up. Was able to fix this by using 1 ul 3066, 0.5 ul 3067, and 0.1 ul 3068. - CLG

WT = 250 bp, floxed = 357 bp Run: 60C x 30 sec, 72C x 30 sec = 35 cycles Mcl-1 loxP F1: GGT TCC CTG TCT CCT TAC TTA CTG TAG Mcl-1 loxP R1: CTC CTA ACC ACT GTT CCT GAC ATC C
 * __Mcl-1-floxed screen__**: Two primers either side of first loxP site yielding different sized bands for WT and floxed alleles reflecting insertion of 107 bp fragment containing loxP.

Neo4 + Neo8 = 600-700 bp Run: 58C – 30 sec, 72C – 1 min = 35 cycles
 * __Neo screen:__** Detects neomycin in TNF family KO (TNFa, TNFRII, LTa) – detects KO allele.
 * Many knockout lines contain Neo so be very cautious when using this to screen and design more specific primers if possible. - ADD

TNFF + TNFR = 200 bp Run: 54C – 30 sec, 72C – 1 min = 35 cycles TNFF: GCA CAG AAA GCA TGA TCC G TNFR: TAG ACA GAA GAG CGT GGT GG
 * __TNFa screen:__** Detects WT allele of TNFa.

__**Atg3 flox screen:**__ 1.atg3 Exon Seq 450 R AACCATAGCCGTGGTGTCTGGTAA 2. atg3 LA seq 4500F CGA TGG CAT CTT ATG CTG AGC AAT G WT: 748bp, Atg3 flox band: 866bp 60 degree for annealing, 1 min. for elongation