Ligation high Ver.2 - Toyobo

www.toyobo.co.jp/e/bioExample 5.TA Cloning using unpurified PCR productsT vector,(50 ng, 25 fmol)was mixed, at different ratios, with unpurified PCR products(500 bp from Taq DNA polymerase). An equal volume of Ligation high Ver.2, 7.5 μl, wasadded and incubated at 16℃ for 30 min. E. coli DH5α competent cells weretransformed using 10 μl of reaction mixture, and cultured on LB/Amp (X-Gal) platesO/N.Relative colony number1201008060402002 1 0.5BlueWhiteAmount of the added PCR products(μl)Fig. 7 TA cloning using unpurified productsColony number on the condition of adding 0.5 μl of the PCR product corresponds to 100.Example 6.Phage vector ligationλZAP ® II (500 ng), EcoRI digested, and pRheo/EcoRI test insert(225 ng, 2.8 kb), weremixed with half or with an equal volume of Ligation high Ver.2, 3.75 or 7.5 μl, andincubated at 16℃ for 30 min. in vitro packaging was performed with 1.5 μl ligationreaction using GIGAPACK ® III(Stratagene) followed by infection of E. coli XL-1 BlueMRF’. The E. coli cells were cultured O/N and plaques counted.Table 2. Reaction conditions and efficiencyDNA soln:Ligation reagent1 : 0.5 1 : 1Efficiency6.3×10 6 5.5×10 6(pfu/μg ZAP ® II)*λZAP ® and GIGAPACK ® are registered trademarks of STRATAGENE.JAPANTOYOBO CO., LTD.Tel(81)-6-6348-3888www.toyobo.co.jp/e/biotech_osaka@toyobo.jpCHINATOYOBO Bio-Technology, CO., LTD.Tel(86)-21-58794900.41405FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[ 5 ] TroubleshootingSymptom Cause SolutionNo or lowcolony numberExcess amount of ligationmixture for transformationUnpurified ligation mixtures should be added at less than10%(V/V) of competent cell suspensions.Cloning efficiency waslowExcess number ofcoloniesVector concentration is lowDNA insert fragmentconcentration is too low.Excess saltsEnds of both vector andinsert are dephosphorylated.Primer dimerInsufficient time for ligationreaction.Insert fragmentconcentration is too low.Self-ligation ofvectorSelf-ligation ofvectorIncrease the vector concentration.(See [4 ] Application data)Increase the DNA insert fragment concentration.(See [4 ] Application data)・DNA fragments should be dissolved in a low salt solution.・Unpurified PCR products should be added at 0.5-1 μl perligation reaction.Dephosphorylated vectors cannot ligate with unphosphorylatedPCR products.When the PCR reaction contains primer dimers or unexpectedPCR products, the desired PCR product should be purified.The reaction can be prolonged for up to 2hr.Increase the insert fragment concentration.(See [4 ] Application data)Use dephosphorylated vector.Use dephosphorylated vector.Freezing of the reagent Storage conditions In the case of -30℃ storage or long-term storage under -20℃,the reagent might freeze. The frozen reagent can be thawed byincubation at room temperature in short time. No decrease inreaction efficiency is observed following 5 freeze-thaw cycles.White precipitates areformedStorage condition In the case of -30℃ storage or long-term storage under -20℃,the reagent might form white precipitates. The precipitatesshould be dissolved prior to use. The precipitates can bedissolved by incubation at room temperature with vortexing atlow speed in short time.JAPANTOYOBO CO., LTD.Tel(81)-6-6348-3888www.toyobo.co.jp/e/biotech_osaka@toyobo.jpCHINATOYOBO Bio-Technology, CO., LTD.Tel(86)-21-58794900.41406FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.