A | expected size 998bp | expected size 998bp | |
components | LHRZ for (BsmBI) | LHRZ for (BsaI) | |
eDA8 (125pg/uL) | 1 | 1 | |
oligo 310 | 1.25 | 1.25 | |
oligo317 | 1.25 | ||
oligo 318 | 1.25 | ||
PrimeStar Premix 2x | 25 | ||
water | up to 50 |
A | B | C | |
310 (F) | LHRZ (BamHI | CCGGATCCCCGTGTAGGTCTACAAACTGG | |
318 (R) | LHRZ (KpnI-BsmBI-AsiSI) | GCGGTACCCGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG | |
317 (R) | LHRZ (KpnI-BsaI-AsiSI) | GGGGTACCGGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG |
Program | time | number of cycles | |
98oC | 10 sec | 33 x | |
55oC | 10sec | ||
68oC | 1min 30 sec | ||
4oC | hold |
A | B | C | D | |
10 x CutSmart | 8 | 8 | 8 | |
BamHI-HF (20u/uL) | 1.5 | 1.5 | 1 | |
KpnI-HF (20u/uL) | 1.5 | 1.5 | 1 | |
PCR LHRZ 310/317 | 50 | |||
PCR LHRZ 310/318 | 50 | |||
pUC19 298 ng/uL | 10 | |||
water | up to 80 -> |
A | 1 | 2 | |
pUC B/K ~100ng/uL) BamHI/KpnI | 1 | 1 | |
T4 DNA ligase | 1 | 1 | |
LHRZ (100ng/ul) B/K(310/317) | 5.5 | ||
10 X buffer | 1 | 1 | |
water | up to 10 -> | ||
LHRZ (100ng/ul)(310/318) B/K | 5.5 |
components | ZeoR | HygR | |
eDA48 (175 (ng/uL) | 1 | ||
eDA78 (125pg/uL) | 1 | ||
oligo 308 | 1.25 | ||
oligo309 | 1.25 | ||
oligo 306 | 1.25 | ||
oligo 307 | 1.25 | ||
PrimeStar Premix 2x | 25 | 25 | |
water | up to 50 | up to 50uL |
A | B | C | |
308 (F) | Zeocin cassette (SalI-BsmBI) overhang GTAG | CTGTCGACCGTCTCAgtagCCCACACACCATAGCTTCAAAATG | |
309(R) | Zeocin cassette (BamHI) | GGGATCCGCAAATTAAAGCCTTCGAGCG | |
306 (F) | HygR (SalI-BsaI) | CCTGTCGACGGTCTCAgtagGACATGGAGGCCCAGAATACCC | |
307 (R) | HygR (BamHI) | GCGGATCCCAGTATAGCGACCAGCATTCACATAC |
components | B | C | D | E | |
10x cutsmart buffer NEB | 8 | 8 | 8 | 8 | |
BamHI-HF (20u/uL) | 1.5 | 1.5 | 1.5 | 1.5 | |
eDA99 70 ng/uL | 30 | ||||
eDA101 81 ng/uL | 30 | ||||
PCR hygR 306/307 | 50 | ||||
PCR zeo 308/309 | 50 | ||||
SalI-HF (20u/uL) | 1.5 | 1.5 | 1.5 | 1.5 | |
water | up to 80 -> |
T4 DNA ligase | 1 | 1 | |
10 X buffer | 1 | 1 | |
eDA101 (S/B) | 3 | ||
zeo 308/309 (S/B) | 5 | ||
eDA99 (S/B) | 3 | ||
hygR 306/307 (S/B) | 5 | ||
water | up to 10 -> |
components | for Gibson 1 | for Gibson 2 | |
water | up to 50 uL | for Gibson 1 | |
Primestar2x | 25 | 25 | |
oligos 327/320 | 1.25/1.25 | ||
oligos 319/326 | 1.25/1.25 | ||
eDA103 (74pg/uL) | 1.5 | 1.5 |
PROGRAM TAK-2.30 | time reaction | cycles | |
98C | 10sec | 33x | |
55C | 10 sec | ||
68C | 2min 30 sec | ||
4C | hold |
A | B | |
Gibson1 319/326 (3.3kb) 40nM 3.3 kb | 1 | |
Gibson2 327/320 (2.05 kb) 40nM | 1 | |
Gibson 2x mix | 2.5 | |
water | 0.5 |
A | B | C | |
AsiSI | 1 | 1 | |
eDA115 326 ng/uL | 3 | ||
eDA103 103 ng/uL | 8 | ||
10 x CutSmart | 3 | 3 | |
water | up to 30 -> |