本橋健研究室（京都産業大学生命科学部）

A series of high-efficiency PCR-cloning vectors and their useful application

We developed three useful vectors for TA-cloning and blunt-end cloning of PCR products (ref. 1). These vectors are available from Addgene (http://www.addgene.org/).

　　　　　　　　　pCRT 　　　　 ID: 120274
　　　　　　　　　pCRZero 　　ID: 120275
　　　　　　　　　pCRZeroT　　ID: 120276

1. T-vectors for TA-cloning can be easily prepared in your laboratory (pCRT and pCRZeroT).
　　　*T-vectors can be easily prepared by digestion with a TypeIIS restriction enzyme.
　　　*Preparation costs of T-vector from pCRT and pCRZeroT are less than 1/100 of commercially available
　　　 T-vectors.
　　　*Background caused by the empty vector can be reduced by the addition of an appropriate restriction
　　　　enzyme other than a TypeIIS restriction enzyme. Background by these empty vectors is lower than
　　　　background by a commercially available T-vector (ref. 1, Table 1).
2. Only positive clones carrying PCR products can form E. coli colonies in blunt-end cloning (pCRZero and pCRZeroT).
　　　*E. coli cells carrying empty vectors cannot form colonies because this system utilizes the E. coli lethal
　　　　ccdB gene.
3. A bifunctional vector can be applied for both TA-cloning and blunt-end cloning (pCRZeroT).
　　　*The vector is designed to be able to be applied to both TA-cloning and blunt-end cloning, according to
　　　 the selection of restriction sites on the vector.

　When these high-efficiency vectors are applied for PCR-cloning, following applications can be utilized.
[Application]
1. PCR products can be directly reacted with non-digested circular vectors using the Golden Gate reaction. This reaction is applicable for both TA-cloning and blunt-end cloning (for pCRZero and pCRZeroT).
　　*E. coli cells carrying empty vectors cannot form colonies because this system utilizes the E. coli lethal
　　　ccdB gene.
2. Unpurified PCR products can be also cloned into linearized vectors for both dA-tailed and blunt-end PCR products.
3. The dA-tailed PCR products amplified by Taq DNA polymerase can be easily cloned using a blunting reaction because the dA-tailed PCR products can be easily blunted by the 3′-5′ exonuclease activity of DNA polymerase (for pCRZero and pCRZeroT).
　　　*In this case, blue/white selection during transformation is not required.
　　　*Only positive clones carrying PCR products can form E. coli colonies

　----------------------------------------

１．TA-cloning
　Ready-to-use T-vectors are generally purchased for the TA-cloning of dA-tailed PCR products amplified by Taq DNA polymerase. T-vectors with a T-overhang can be easily prepared from pCRT and pCRZeroT vectors in your laboratory through a simple digestion with the type IIS restriction enzyme XcmI. Moreover, addition of NheI (for pCRT) or XmaI (for pCRZeroT) at the same time as XcmI, can reduce background colony-formation.

Protocol for TA-cloning
　　　1. Digest pCRT or pCRZeroT with XcmI.
　　　　　　　　Reaction (for pCRT):
　　　　　　　　　　　　　pCRT 5 µg
　　　　　　　　　　　　　XcmI 20 units
　　　　　　　　　　　　　NheI 15 units
　　　　　　　　　　　　　Buffer NEB 2.1 total 50 µL
　　　　　　　　　　　　　37°C >120 min

　　　　　　　　Reaction (for pCRZeroT):
　　　　　　　　　　　　　pCRZeroT 5 µg
　　　　　　　　　　　　　XcmI 20 units
　　　　　　　　　　　　　XmaI 10 units
　　　　　　　　　　　　　Buffer NEB 2.1 total 50 µL
　　　　　　　　　　　　　37°C, >120 min
　　　　　　　　* To reduce background colony-formation, NheI (for pCRT) or XmaI (for pCRZeroT) should be
　　　　　　　　　added at the same time as XcmI.

　　　2. Purify DNA fragments with a gel/PCR extraction kit (Nippon Genetics)
　　　　　　　　Elution volume: 50 µL
　　　　　　　　* This procedure does not require separation by agarose gel electrophoresis.
　　　　　　　　* Prepare to 50－100 ng/µL, then store at -20°C.

　　　3. Ligate the dA-tailed PCR products amplified by Taq DNA polymerase into pCRT or pCRZeroT.
　　　　　　　　Ligation:
　　　　　　　　　　　Linearized pCRT (or pCRZeroT) 1 µL (50－100 ng)
　　　　　　　　　　　PCR products (purified) 1/20 vol. of PCR products
　　　　　　　　　　　　　　(or PCR products (unpurified) 1/20 vol. of PCR products)
　　　　　　　　　　　　　　　　　at 16°C for 30 min (or 4°C　O/N)
　　　　　　　　* The E. coli colony number is sufficient following ligation with 0.5 µL of vector (50－100 ng/µL)
　　　　　　　　　 when the transformation efficiency of E. coli competent cells is high (~10⁷ CFUs/ug pUC19
　　　　　　　　　 DNA).

　　　4. Transform E. coli competent cells.

　----------------------------------------

２．Blunt-end cloning　　　
　Highly processive DNA polymerases, such as Phusion DNA polymerase, KOD DNA polymerase, and Prime STAR DNA polymerase, have recently been used for high-fidelity PCR. Cloning their PCR products requires blunt-end cloning. Blunt-end cloning is generally less efficient than cohesive-end cloning. For this reason, several researchers have used the TA-cloning system after the addition of dA to the 3′-end of blunt-end PCR products. However, if you use pCRZero or pCRZeroT, blunt-end cloning can be easily used to directly clone blunt-end PCR products because this system involves positive selection, and clones with empty vectors do not appear as E. coli colonies because they express the lethal ccdB gene. This system is very useful.

Protocol for blunt-end cloning
　　　1. Prepare pCRZero and pCRZeroT.
　E. coli cells carrying empty pCRZero and pCRZeroT vectors express the lethal ccdB gene. For the reason, the preparation of empty vectors of pCRZero and pCRZeroT requires E. coli ccdB-resistant strains. The JM109, XL10-Gold, and NEB Turbo strains containing the F-plasmid can be used as ccdB-resistant strains.
　　　　　　　　* If growth of E. coli cells carrying pCRZero and pCRZeroT is not sufficient even when using
　　　　　　　　　 ccdB-resistant strains, we recommend following options.
　　　　　　　　　　　　　① Use of ccdB-resistant strains having lacI^q gene.
　　　　　　　　　　　　　② Addition of glucose (final 1%) to both LB-plate and LB liquid medium.
　　　　　　　　　　　　　③ Scale-up of LB liquid medium to 4-fold volume.

　　　2. Linearize pCRZero or pCRZeroT for blunt-end cloning.
　　　　　　　　2-1. Digest pCRZero with EcoRV.
　　　　　　　　　　　　　Reaction (for pCRZero):
　　　　　　　　　　　　　　　pCRZero 5 µg
　　　　　　　　　　　　　　　EcoRV 30 units
　　　　　　　　　　　　　　　Buffer H (Takara Bio) total 50 µL
　　　　　　　　　　　　　　　37°C, >120 min

　　　　　　　　　　　Digest pCRZeroT with SmaI.
　　　　　　　　　　　　　Reaction (for pCRZeroT):
　　　　　　　　　　　　　　　pCRZeroT 5 µg
　　　　　　　　　　　　　　　SmaI 20 units
　　　　　　　　　　　　　　　Buffer T + BSA (Takara Bio) total 50 µL
　　　　　　　　　　　　　　　30°C, >120 min

　　　　　　　　2-2. Purify DNA fragments with a gel/PCR extraction kit (Nippon Genetics)
　　　　　　　　　　　Elution volume: 50 µL
　　　　　　　　　　　　　* This procedure does not require separation by agarose gel electrophoresis.
　　　　　　　　　　　　　* Prepare to 50－100 ng/µL, then store at -20°C.

　　　3. Screen positive clones using the E. coli lethal ccdB gene for blunt-end cloning of PCR products.
　　　　　　　　3-1. Ligate the blunt-end PCR products to blunt-end pCRZero or pCRZeroT.
　　　　　　　　　　　Ligation:
　　　　　　　　　　　　　Linearized pCRZero (or pCRZeroT) 1 µL (50－100 ng)
　　　　　　　　　　　　　PCR products (purified) 1/20 vol. of PCR products
　　　　　　　　　　　　　　　　(or PCR products (unpurified) 1/20 vol. of PCR products)
　　　　　　　　　　　　　　　　　　　at 16°C for 30 min (or 4°C　O/N)
　　　　　　　　　　　　　* The E. coli colony number is sufficient following ligation with 0.5 µL of vector (50－100
　　　　　　　　　　　　　　 ng/µL) when the transformation efficiency of E. coli competent cells is high (~10⁷
　　　　　　　　　　　　　　 CFUs/ug pUC19 DNA).

　　　　　　　　3-2. Transform E. coli ccdB-sensitive competent cells.
　　　　　　　　　　　　　* The positive selection of plasmids carrying PCR products requires using E. coli
　　　　　　　　　　　　　　 ccdB-sensitive strains. The DH5-alpha, TOP10, Mach1-T1R, NEB 5-alpha, and NEB
　　　　　　　　　　　　　　 10-beta strains can be used as ccdB-sensitive strains.

　　　　　　　　3-3. Several colonies should be screened by colony-PCR, and insertion of PCR products should
　　　　　　　　　　　 be confirmed.

[Application 1] Direct cloning of PCR products into non-digested circular vectors using the Golden Gate Reaction.
　PCR products can be directly reacted with the two non-digested circular vectors pCRZero and pCRZeroT containing the E. coli lethal ccdB gene using the Golden Gate reaction. This reaction is applicable for both TA-cloning and blunt-end cloning. Using non-digested circular plasmids results in the same cloning efficiency as when with using a linearized vector.

　　　　　　　　Reaction 1 (for dA-tailed PCR products):
　　　　　　　　　　　　　pCRZeroT (non-cut) 50 ng
　　　　　　　　　　　　　dA-tailed PCR products 5:1 (i:v molar ratio)
　　　　　　　　　　　　　XcmI 10 units
　　　　　　　　　　　　　XmaI 5 units
　　　　　　　　　　　　　Quick Ligase 0.5 µL
　　　　　　　　　　　　　Buffer CutSmart (2×, final)
　　　　　　　　　　　　　ATP 1 mM (final) total 10 µL

　　　　　　　　Reaction 2 (for blunt-end PCR products):
　　　　　　　　　　　　　pCRZeroT (non-cut) 50 ng
　　　　　　　　　　　　　blunt-end PCR products 5:1 (i:v molar ratio)
　　　　　　　　　　　　　SmaI 10 units
　　　　　　　　　　　　　Quick Ligase 0.5 µL
　　　　　　　　　　　　　Buffer CutSmart (2×, final)
　　　　　　　　　　　　　ATP 1 mM (final) total 10 µL

　　　　　　　　Reaction 3 (for blunt-end PCR products):
　　　　　　　　　　　　　pCRZero (non-cut) 50 ng
　　　　　　　　　　　　　blunt-end PCR products 5:1 (i:v molar ratio)
　　　　　　　　　　　　　SmaI 15 units
　　　　　　　　　　　　　Quick Ligase 0.5 µL
　　　　　　　　　　　　　Buffer CutSmart (2×, final)
　　　　　　　　　　　　　ATP 1 mM (final) total 10 µL

　　　　　　　　(37°C -1 min + 16°C -1 min) × 30 times and 80°C -5 min

　　　　　　　　*It is required that the PCR products contain no restriction sites used for vector linearization.
　　　　　　　　*When the insert:vector is set up at the appropriate molar ratio, non-digested circular plasmids
　　　　　　　　　 have the same cloning efficiency as linearized vectors.
　　　　　　　　　　　　　See ref. 1, Table 3 and Supplementary Figure S1
　　　　　　　　　　　　　Reagent, Quick Ligation Kit (M2200S, NEB)

[Application 2] Cloning of unpurified PCR products
　E. coli cells carrying an empty plasmid of pCRZeroT cannot form colonies because the plasmid encodes the lethal ccdB gene. Both purified and unpurified PCR products can be cloned efficiently in dA-tailed and blunt-end PCR products.
　　　　　　　　　　　　　See ref. 1, Table 4

[Application 3] Blunt-end cloning by blunting of the dA-tailed PCR products
　The dA-tailed PCR products amplified by Taq DNA polymerase are cloned by TA-cloning because of the addition of dA at the 3′-end of the products. Blue/white selection with IPTG/X-gal is generally used in TA-cloning. In contrast, positive selection can be used with both pCRZero and pCRZeroT carrying the E. coli lethal ccdB gene when dA-tailed PCR products are blunted by KOD DNA polymerase. Blue/white selection is not required in the ccdB-dependent positive selection system. The blunting of dA-tailed PCR products can be easily performed.
　　　　　　　　* “Blunting of dA-tailed PCR products” refers to the supplement.^*1
　　　　　　　　　　　　　See ref. 1, Table 5

^*1Supplement
　　　Blunting of dA-tailed PCR products
　　　　　　　　KOD DNA polymerase with 3′-5′ exonuclease activity can easily blunt the dA-overhang end of
　　　　　　　　the dA-tailed PCR products that are amplified by Taq DNA polymerase.
　　　　　　　　　　　　　1. Solution after PCR reaction (20－50 µL).
　　　　　　　　　　　　　　　　*Solution after PCR reaction does not require any purification step.
　　　　　　　　　　　　　2. Add 1.25 U of KOD DNA polymerase.
　　　　　　　　　　　　　　　　*Add dNTPs (final 0.2 mM) when the dNTPs concentration is insufficient.
　　　　　　　　　　　　　3. Incubate at 72°C for 2－30 min (minimum 2 min)
　　　　　　　　　　　　　4. Apply the following steps (ligation (or purification))
　　　　　　　　　　　　　　　　*Generally, 1－5 µL of reaction mixture is directly applied to the ligation reaction.
　　　　　　　　　　　　　See ref. 1, Table 5
　　　　　　　　　　　　　Reagent, KOD DNA polymerase (250 units, KOD-101, Toyobo)

----------------------------------------
You can obtain the pCRT, pCRZero, and pCRZeroT plasmids from Addgene (http://www.addgene.org/).
More information is provided in the following reference.
Please cite the following reference if you use these plasmids for your scientific work.

ref. 1 Ken Motohashi

"A novel series of high-efficiency vectors for TA cloning and blunt-end cloning of PCR products"
　Sci. Rep. 9, 6417 (2019)




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We developed three useful vectors for TA-cloning and blunt-end cloning of PCR products (ref. 1). These vectors are available from Addgene (http://www.addgene.org/).
pCRT 　　　　 ID: 120274 　　　　　　　　　pCRZero 　　ID: 120275 　　　　　　　　　pCRZeroT　　ID: 120276

1. T-vectors for TA-cloning can be easily prepared in your laboratory (pCRT and pCRZeroT). 　　　T-vectors can be easily prepared by digestion with a TypeIIS restriction enzyme. 　　　Preparation costs of T-vector from pCRT and pCRZeroT are less than 1/100 of commercially available 　　　 T-vectors. 　　　Background caused by the empty vector can be reduced by the addition of an appropriate restriction 　　　　enzyme other than a TypeIIS restriction enzyme. Background by these empty vectors is lower than 　　　　background by a commercially available T-vector (ref. 1, Table 1). 2. Only positive clones carrying PCR products can form E. coli* colonies in blunt-end cloning (pCRZero and pCRZeroT). 　　　*E. coli cells carrying empty vectors cannot form colonies because this system utilizes the E. coli lethal 　　　　ccdB gene. 3. A bifunctional vector can be applied for both TA-cloning and blunt-end cloning (pCRZeroT). 　　　The vector is designed to be able to be applied to both TA-cloning and blunt-end cloning, according to 　　　 the selection of restriction sites on the vector. 　When these high-efficiency vectors are applied for PCR-cloning, following applications can be utilized. [Application] 1. PCR products can be directly reacted with non-digested circular vectors using the Golden Gate reaction. This reaction is applicable for both TA-cloning and blunt-end cloning (for pCRZero and pCRZeroT). 　　E. coli cells carrying empty vectors cannot form colonies because this system utilizes the E. coli lethal 　　　ccdB gene. 2. Unpurified PCR products can be also cloned into linearized vectors for both dA-tailed and blunt-end PCR products. 3. The dA-tailed PCR products amplified by Taq DNA polymerase can be easily cloned using a blunting reaction because the dA-tailed PCR products can be easily blunted by the 3′-5′ exonuclease activity of DNA polymerase (for pCRZero and pCRZeroT). 　　　In this case, blue/white selection during transformation is not required. 　　　Only positive clones carrying PCR products can form E. coli colonies

----------------------------------------
１．TA-cloning 　Ready-to-use T-vectors are generally purchased for the TA-cloning of dA-tailed PCR products amplified by Taq DNA polymerase. T-vectors with a T-overhang can be easily prepared from pCRT and pCRZeroT vectors in your laboratory through a simple digestion with the type IIS restriction enzyme XcmI. Moreover, addition of NheI (for pCRT) or XmaI (for pCRZeroT) at the same time as XcmI, can reduce background colony-formation.
Protocol for TA-cloning 　　　1. Digest pCRT or pCRZeroT with XcmI. 　　　　　　　　Reaction (for pCRT): 　　　　　　　　　　　　　pCRT 5 µg 　　　　　　　　　　　　　XcmI 20 units 　　　　　　　　　　　　　NheI 15 units 　　　　　　　　　　　　　Buffer NEB 2.1 total 50 µL 　　　　　　　　　　　　　37°C >120 min 　　　　　　　　Reaction (for pCRZeroT): 　　　　　　　　　　　　　pCRZeroT 5 µg 　　　　　　　　　　　　　XcmI 20 units 　　　　　　　　　　　　　XmaI 10 units 　　　　　　　　　　　　　Buffer NEB 2.1 total 50 µL 　　　　　　　　　　　　　37°C, >120 min 　　　　　　　　* To reduce background colony-formation, NheI (for pCRT) or XmaI (for pCRZeroT) should be 　　　　　　　　　added at the same time as XcmI. 　　　2. Purify DNA fragments with a gel/PCR extraction kit (Nippon Genetics) 　　　　　　　　Elution volume: 50 µL 　　　　　　　　* This procedure does not require separation by agarose gel electrophoresis. 　　　　　　　　* Prepare to 50－100 ng/µL, then store at -20°C. 　　　3. Ligate the dA-tailed PCR products amplified by Taq DNA polymerase into pCRT or pCRZeroT. 　　　　　　　　Ligation: 　　　　　　　　　　　Linearized pCRT (or pCRZeroT) 1 µL (50－100 ng) 　　　　　　　　　　　PCR products (purified) 1/20 vol. of PCR products 　　　　　　　　　　　　　　(or PCR products (unpurified) 1/20 vol. of PCR products) 　　　　　　　　　　　　　　　　　at 16°C for 30 min (or 4°C　O/N) 　　　　　　　　* The E. coli colony number is sufficient following ligation with 0.5 µL of vector (50－100 ng/µL) 　　　　　　　　　 when the transformation efficiency of E. coli competent cells is high (~10⁷ CFUs/ug pUC19 　　　　　　　　　 DNA). 　　　4. Transform E. coli competent cells.
----------------------------------------
２．Blunt-end cloning　　　　Highly processive DNA polymerases, such as Phusion DNA polymerase, KOD DNA polymerase, and Prime STAR DNA polymerase, have recently been used for high-fidelity PCR. Cloning their PCR products requires blunt-end cloning. Blunt-end cloning is generally less efficient than cohesive-end cloning. For this reason, several researchers have used the TA-cloning system after the addition of dA to the 3′-end of blunt-end PCR products. However, if you use pCRZero or pCRZeroT, blunt-end cloning can be easily used to directly clone blunt-end PCR products because this system involves positive selection, and clones with empty vectors do not appear as E. coli colonies because they express the lethal ccdB gene. This system is very useful.
Protocol for blunt-end cloning 　　　1. Prepare pCRZero and pCRZeroT. 　E. coli cells carrying empty pCRZero and pCRZeroT vectors express the lethal ccdB gene. For the reason, the preparation of empty vectors of pCRZero and pCRZeroT requires E. coli ccdB-resistant strains. The JM109, XL10-Gold, and NEB Turbo strains containing the F-plasmid can be used as ccdB-resistant strains. 　　　　　　　　* If growth of E. coli cells carrying pCRZero and pCRZeroT is not sufficient even when using 　　　　　　　　　 ccdB-resistant strains, we recommend following options. 　　　　　　　　　　　　　① Use of ccdB-resistant strains having lacI^q gene. 　　　　　　　　　　　　　② Addition of glucose (final 1%) to both LB-plate and LB liquid medium. 　　　　　　　　　　　　　③ Scale-up of LB liquid medium to 4-fold volume. 　　　2. Linearize pCRZero or pCRZeroT for blunt-end cloning. 　　　　　　　　2-1. Digest pCRZero with EcoRV. 　　　　　　　　　　　　　Reaction (for pCRZero): 　　　　　　　　　　　　　　　pCRZero 5 µg 　　　　　　　　　　　　　　　EcoRV 30 units 　　　　　　　　　　　　　　　Buffer H (Takara Bio) total 50 µL 　　　　　　　　　　　　　　　37°C, >120 min 　　　　　　　　　　　Digest pCRZeroT with SmaI. 　　　　　　　　　　　　　Reaction (for pCRZeroT): 　　　　　　　　　　　　　　　pCRZeroT 5 µg 　　　　　　　　　　　　　　　SmaI 20 units 　　　　　　　　　　　　　　　Buffer T + BSA (Takara Bio) total 50 µL 　　　　　　　　　　　　　　　30°C, >120 min 　　　　　　　　2-2. Purify DNA fragments with a gel/PCR extraction kit (Nippon Genetics) 　　　　　　　　　　　Elution volume: 50 µL 　　　　　　　　　　　　　* This procedure does not require separation by agarose gel electrophoresis. 　　　　　　　　　　　　　* Prepare to 50－100 ng/µL, then store at -20°C. 　　　3. Screen positive clones using the E. coli lethal ccdB gene for blunt-end cloning of PCR products. 　　　　　　　　3-1. Ligate the blunt-end PCR products to blunt-end pCRZero or pCRZeroT. 　　　　　　　　　　　Ligation: 　　　　　　　　　　　　　Linearized pCRZero (or pCRZeroT) 1 µL (50－100 ng) 　　　　　　　　　　　　　PCR products (purified) 1/20 vol. of PCR products 　　　　　　　　　　　　　　　　(or PCR products (unpurified) 1/20 vol. of PCR products) 　　　　　　　　　　　　　　　　　　　at 16°C for 30 min (or 4°C　O/N) 　　　　　　　　　　　　　* The E. coli colony number is sufficient following ligation with 0.5 µL of vector (50－100 　　　　　　　　　　　　　　 ng/µL) when the transformation efficiency of E. coli competent cells is high (~10⁷ 　　　　　　　　　　　　　　 CFUs/ug pUC19 DNA). 　　　　　　　　3-2. Transform E. coli ccdB-sensitive competent cells. 　　　　　　　　　　　　　* The positive selection of plasmids carrying PCR products requires using E. coli 　　　　　　　　　　　　　　 ccdB-sensitive strains. The DH5-alpha, TOP10, Mach1-T1R, NEB 5-alpha, and NEB 　　　　　　　　　　　　　　 10-beta strains can be used as ccdB-sensitive strains. 　　　　　　　　3-3. Several colonies should be screened by colony-PCR, and insertion of PCR products should 　　　　　　　　　　　 be confirmed.
[Application 1] Direct cloning of PCR products into non-digested circular vectors using the Golden Gate Reaction. 　PCR products can be directly reacted with the two non-digested circular vectors pCRZero and pCRZeroT containing the E. coli lethal ccdB gene using the Golden Gate reaction. This reaction is applicable for both TA-cloning and blunt-end cloning. Using non-digested circular plasmids results in the same cloning efficiency as when with using a linearized vector.

Reaction 1 (for dA-tailed PCR products): 　　　　　　　　　　　　　pCRZeroT (non-cut) 50 ng 　　　　　　　　　　　　　dA-tailed PCR products 5:1 (i:v molar ratio) 　　　　　　　　　　　　　XcmI 10 units 　　　　　　　　　　　　　XmaI 5 units 　　　　　　　　　　　　　Quick Ligase 0.5 µL 　　　　　　　　　　　　　Buffer CutSmart (2×, final) 　　　　　　　　　　　　　ATP 1 mM (final) total 10 µL 　　　　　　　　Reaction 2 (for blunt-end PCR products): 　　　　　　　　　　　　　pCRZeroT (non-cut) 50 ng 　　　　　　　　　　　　　blunt-end PCR products 5:1 (i:v molar ratio) 　　　　　　　　　　　　　SmaI 10 units 　　　　　　　　　　　　　Quick Ligase 0.5 µL 　　　　　　　　　　　　　Buffer CutSmart (2×, final) 　　　　　　　　　　　　　ATP 1 mM (final) total 10 µL 　　　　　　　　Reaction 3 (for blunt-end PCR products): 　　　　　　　　　　　　　pCRZero (non-cut) 50 ng 　　　　　　　　　　　　　blunt-end PCR products 5:1 (i:v molar ratio) 　　　　　　　　　　　　　SmaI 15 units 　　　　　　　　　　　　　Quick Ligase 0.5 µL 　　　　　　　　　　　　　Buffer CutSmart (2×, final) 　　　　　　　　　　　　　ATP 1 mM (final) total 10 µL 　　　　　　　　(37°C -1 min + 16°C -1 min) × 30 times and 80°C -5 min 　　　　　　　　It is required that the PCR products contain no restriction sites used for vector linearization. 　　　　　　　　When the insert:vector is set up at the appropriate molar ratio, non-digested circular plasmids 　　　　　　　　　 have the same cloning efficiency as linearized vectors. 　　　　　　　　　　　　　See ref. 1, Table 3 and Supplementary Figure S1 　　　　　　　　　　　　　Reagent, Quick Ligation Kit (M2200S, NEB)
[Application 2] Cloning of unpurified PCR products 　E. coli cells carrying an empty plasmid of pCRZeroT cannot form colonies because the plasmid encodes the lethal ccdB gene. Both purified and unpurified PCR products can be cloned efficiently in dA-tailed and blunt-end PCR products. 　　　　　　　　　　　　　See ref. 1, Table 4
[Application 3] Blunt-end cloning by blunting of the dA-tailed PCR products 　The dA-tailed PCR products amplified by Taq DNA polymerase are cloned by TA-cloning because of the addition of dA at the 3′-end of the products. Blue/white selection with IPTG/X-gal is generally used in TA-cloning. In contrast, positive selection can be used with both pCRZero and pCRZeroT carrying the E. coli lethal ccdB gene when dA-tailed PCR products are blunted by KOD DNA polymerase. Blue/white selection is not required in the ccdB-dependent positive selection system. The blunting of dA-tailed PCR products can be easily performed. 　　　　　　　　* “Blunting of dA-tailed PCR products” refers to the supplement.^1 　　　　　　　　　　　　　See ref. 1, Table 5 ^1Supplement 　　　Blunting of dA-tailed PCR products 　　　　　　　　KOD DNA polymerase with 3′-5′ exonuclease activity can easily blunt the dA-overhang end of 　　　　　　　　the dA-tailed PCR products that are amplified by Taq DNA polymerase. 　　　　　　　　　　　　　1. Solution after PCR reaction (20－50 µL). 　　　　　　　　　　　　　　　　Solution after PCR reaction does not require any purification step. 　　　　　　　　　　　　　2. Add 1.25 U of KOD DNA polymerase. 　　　　　　　　　　　　　　　　Add dNTPs (final 0.2 mM) when the dNTPs concentration is insufficient. 　　　　　　　　　　　　　3. Incubate at 72°C for 2－30 min (minimum 2 min) 　　　　　　　　　　　　　4. Apply the following steps (ligation (or purification)) 　　　　　　　　　　　　　　　　*Generally, 1－5 µL of reaction mixture is directly applied to the ligation reaction. 　　　　　　　　　　　　　See ref. 1, Table 5 　　　　　　　　　　　　　Reagent, KOD DNA polymerase (250 units, KOD-101, Toyobo)
---------------------------------------- You can obtain the pCRT, pCRZero, and pCRZeroT plasmids from Addgene (http://www.addgene.org/). More information is provided in the following reference. Please cite the following reference if you use these plasmids for your scientific work.
ref. 1	Ken Motohashi
	"A novel series of high-efficiency vectors for TA cloning and blunt-end cloning of PCR products" 　Sci. Rep. 9, 6417 (2019)