GV pXT19-T Vector

Cat #GT1502-20T

GV pXT19-T Vector is a high efficient cloning vector for PCR products. It is convertedfrom pUC19.For most of the heat resistance DNA polymerases producing "A" at 3’ end, the product can greatly improveclone efficiency. The vector has the same function as pUC19. It has β-galactosidase with high expression activity and is ready for white/blue screening after cloning. Dark blue clone is visible for overnight cultivation the night is dark blue bacterial colonies training. Meanwhile, the vector has ampr resistance gene for screening the recombinant.

一、Component and storage

pXT19-T Vector(50 ng/μl)

20 μlx1

Control Insert(50 ng/μl)  

10 μlx1

2×Solution I

75 μlx2

Primer M13(-47)(10μM)  

50 μl

Primer RV-M(10μM) 

50 μl

Storage: -20℃

Application

          TA Cloning for PCR products

        Sequencing with M13 Primers for clones

二、GV pXT19-T Vector structure

GV pXT19-T Vector sequence reference points

Cloning site

441

BcaBEST Sequencing Primer M13-47 binding site

352-375

BcaBEST Sequencing Primer RV-M binding site

504-527

LacZ operator

146-495

ColE1 ori

893-1481

Ampr

1652-2512

 

三、Protocol

1、   Set up ligation reactions as described below at 0.2ml tubes, mix well by pipetting. 

pXT19-T Vector(50 ng/μl)

1μl

Insert DNA 

0.1pmol—0.3pmol

2×Solution I

5μl

ddH2O

up to 10μl

2、   Incubate the reactions 30min at 16℃, extend the time for products longer than 1kb.

      Notes: ①Incubate at room temperature will reduce ligation efficiency slightly.

            ②Incubate for 5 min will reduce ligation efficiency slightly.

            ③For cloning products longer than 2kb, please incubate the reaction for more hours or overnight at 16℃.

3、   Add 10μl ligation reaction to 100μl DH5a competent cells. Place 30min in an ice bath.

4、   Heat-shock the cells for 90s at 42℃. Immediately return the tubes to ice for 1-2min.

5、   Add 900μl LB(or SOC)medium. Incubate for 1h at 37℃ with shaking(~160rpm).

6、   Centrifuge 1min at 4000rpm. Suspend cells with 100μl supernatant.

7、   Plate cells onto X-Gal/IPTG/Amp/LB plates. Incubate overnight at 37℃.

8、   Select white colonies to identify insert.

四、Notes

1、   Dissolve2×Solution in an ice bath。

2、   Ligation solution for transforming is less than 20μl. For transforming large quantities DNA or electrotransformation, precipitate the ligation solution with alcohol to purify DNA.

3、   Incubate the reactions at temperature below 25℃, because high temperature is disadvantage for forming circle DNA. Please extend ligation time for few hours when ligation efficiency is low.

4、   Do positive control experiment when necessary. According to the experimental protocol, clone the Control insert in the kit can get positive rate over 90%. The kit provides positive control experiment for 10 times.

5、   2×Solution I in the kit can be also used in ligation reaction at vector construction experiments.

五、Attentions

1、   Component cell

      The pXT19-T Vector is derived from pUC19 and is suitable for component cell such as DH5α, JM109.

      Please use high efficiency component cell (DH5α, JM109 etc) with heat shock method to get enough positive clones. For blue/white screening, the host cell should be LacZ△M15. The w fragment encode by F′ can combine with LacZ α peptide encoded by vector DNA, and show β-galactosidase enzymatic activity (α-complementarity).

2、   Insert DNA

      Insert DNA should be purified before ligation reaction to avoid polluting by impurity. To purify the insert DNA, you can use GV-High Efficiency Agarose Gel DNA Purification Kit(Cat #:GV-GX-50) or GV-High Efficiency DNA Purification Kit(Cat #:GV-PCR-P-50).

3、   Dosage of the insert DNA

    The mol rate of Vector DNA︰Insert DNA is 12~1︰10 according to experiment condition. Insert DNA dosage can be calculated as follows:

Insert DNA dosage (ng) = nmol Number×660×bp Number

    The vector in kit 1μl (50ng) is about 0.03pmol.

4、   Detection for positive clones

   When DNA insert to pXT19-T Vector, the β-galactosidase expression will be destroyed. Recombinant shows white clones on X-Gal/IPTG/Amp/LB plates. Using PCR to detect positive clones, you can use primer provided by the kit: M13 (-47) (10 μM) and RV-M (10 μM).

5、   Transformation efficiency

          Transformant number = (cfu×dilution ratio×stock solution volume)/solution volume for plate

      Transformation efficiency = transformant number / clones of plasmid DNA (1µg superhelix plasmid DNA)

 

 

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