Worm Strain: RG3151
Allele Name: ve651
Gene: T20B5.2
Parent Strain: N2
Phenotype: Homozygotes are unhealthy.

Genotype: +/szT1 [lon-2(e678) umnIs61] I; T20B5.2(ve651[LoxP + myo-2p::GFP::unc-54 3' UTR + rps-27p::neoR::unc-54 3' UTR + LoxP])/szT1 X.

Description: umnIs61 [myo-2p::mKate2 + NeoR, X: 15420938 (intergenic)] I. Homozygotes are unhealthy. Deletion of 5228 bp with Calarco/Colaiacovo selection cassette conferring myo-2 GFP and G418 resistance inserted at break in parental strain N2. Heterozygotes are wild-type GFP+ mKate2+, and segregate wild-type GFP+ mKate2+, GFP+ non-mKate2 sickly adults (ve651 homozygotes), Lon non-GFP mKate2+ males (szT1 hemizygotes), and dead eggs (szT1 homozygotes and aneuploids). Maintain by picking wild-type GFP+ mKate2+. Left flanking Sequence: aaggggagggaaaacagttgaggacttttg ; Right flanking sequence: GAATGCGCATACTTGATGGAAAACCCGCTC. sgRNA #1: aacagttgaggacttttggt; sgRNA #2: ATCAAGTATGCGCATTCGCT. Please reference Au et al., G3 9(1): 135-144 2019 in any work resulting from use of this mutation.

T20B5.2 sgRNA #1: aacagttgaggacttttggt
T20B5.2 sgRNA #2: ATCAAGTATGCGCATTCGCT

Left Flanking Sequence: aaggggagggaaaacagttgaggacttttg
Right Flanking Sequence: GAATGCGCATACTTGATGGAAAACCCGCTC

Deletion: 5228 bp

Wildtype DNA File: T20B5.2_Wildtype
Insertion DNA File: T20B5.2_Insertion
Plasmid: Guides: pMLV-FL1 (T20B5.2 sgRNA #1), pMLV-FL2 (T20B5.2 sgRNA #2)
Plasmid: Template: pMLV-HA-FL (T20B5.2 repair)

QC: PPPP

T20B5.2 Primers:
T20B5.2-FGP: aatgggaacaagccgtcact
T20B5.2-RGP: tgacaaaggagagagctggc
T20B5.2-IFGP: aaagggcagtgagacaaggg
T20B5.2-IRGP: TGACAGGTCGGACTGGTGAT
loxPmyo2NeoR-FCP: CTGCATGCGTCGACATAACT
loxPmyo2NeoR-RCP: CGTAGAGCTCGGTACCTCGT

Expected PCR results:
Primer Pair				WT		In/Del
T20B5.2-FGP	T20B5.2-RGP		3934 bp		-
T20B5.2-FGP	T20B5.2-RGP		2948 bp		-
T20B5.2-FGP	loxPMyo2NeoR-RCP	-		1107 bp
T20B5.2-RGP	loxPMyo2NeoR-FCP	-		1051 bp

PCR Parameters: (NEB Quick-load 2x Taq Master Mix)
(1) 95C/30 sec
(2) 95C/30 sec
(3) 58C/30 sec
(4) 68C/4:00 min
(5) To step (2) x 34 times
(6) 68C/5:00
