30 rxn

The pKRX-T vector was made by cutting the pKRX plasmid which was developed by cloning a cassette containing two Xcm I recognition sites between EcoR I and Sal I sites of pBluescript II SK(+), after digestion by Xcm I, generated single 3' thymidine overhangs on both ends of the vector. These single 3'-T overhangs at the insertion site greatly improve the efficiency of ligation of PCR products into the plasmids by preventing recircularization of the vectors and providing compatible overhangs for PCR products generated by certain thermostable polymerases such as Taq and Tth which often add a single deoxyadenosine to the 3'-end of the amplified fragments.

pKRX-T Vector Structure

1. Sequence of pKRX-T Vector Promoters and MCS

Product Components

            pKRX-T Vector(30ng/μl)                           30μl × 1

           Control Insert DNA(10ng/μl)                 30μl × 1

           T7 Promoter Primer                                  1nmol × 1

           T3 Promoter Primer                                  1nmol × 1

1.  When molar ratios of the Control Insert DNA (500bp) to the vectors is 1:1 to 50:1, > 90% of the whole transformants are recombinant colonies.

2. After cloning the Control Insert DNA, sequencing to confirm MCS and thymidines.

Protocol

A. PCR Product Cloning

1.  PCR product Purification and Quantification

The PCR product to be ligated should be gel-purified and determine the amount of DNA by comparing with markers.

2.  LIgation

Set up ligation reactions as described below.

         10×T4 DNALigase Buffer                          1 μl

         pKRX-T（30ng/μl）                                   1 μl

         Gel-purified PCR Product                         X μl*

         T4 DNA Ligase                                           1~3 Weiss Units

         ddH₂O                                                          up to10 μl

* The best molar ratios of the PCR product to the vector are 2:1~10:1.

Mix the reactions by pipetting. Incubate the reactions 3 hour or overnight at 16℃. Longer incubation times will increase the number of transformants. (Generally; incubation overnight at 16℃ will produce the maximum number of transformants.)

3.  Transformations

1) Thaw 100μl competent cells on ice.

2) Add 5μl ligation reaction，swirl tube, incubate on ice for 30 minutes.

3) Heat-shock the cells for 90 seconds in a water bath at exactly 42℃ (Do not shake). Immediately return the tubes to ice for 2 minutes.

4) Add 900µl room temperature LB medium to the tubes. Incubate for 1 hour at 37℃ with shaking (~150rpm).

5) Plate 100µl of each transformation culture onto duplicate LB/ampicillin plates or LB/ampicillin/IPTG/X-Gal plates. If a higher number of colonies is desired, the cells may be pelleted by centrifugation at 1,000 × g for 10 minutes, resuspended in 200µl of SOC medium, and 100µl plated on each of two plates.

6) Incubate the plates overnight (16~24 hours) at 37℃.

4.  Identification of recombinant colonies

Take cells from fresh plates (or take white cells from LB/ampicillin/IPTG/X-Gal plates), culture in LB/ampicillin medium overnight at 37℃, isolate plasmid. Identify the recombinant colonies by double digestion with two different restriction endonucleases or PCR with T7/T3 promoter primers. Colony PCR Method see B．Control Insert DNA Cloning

B.  Control Insert DNA Cloning

1.  LIgation

Set up ligation reactions as described below.

          10×T4 DNA Ligase Buffer                         1 μl

          pKRX-T(30ng/μl)                                         1 μl

          Control Insert DNA(10ng/μl)                     2~3 μl

          T4 DNA Ligase                                           1~3 Weiss Units

          ddH₂O                                                           up to10 μl

Mix the reactions by pipetting. Incubate the reactions 3 hour or overnight at 16℃. Longer incubation times will increase the number of transformants. (Generally; incubation overnight at 16℃ will produce the maximum number of transformants.)

2.  Transformations

1) Thaw 100μl competent cells on ice.

2) Add 5μl ligation reaction，swirl tube, incubate on ice for 30 minutes.

3) Heat-shock the cells for 90 seconds in a water bath at exactly 42℃ (Do not shake). Immediately return the tubes to ice for 2 minutes.

4) Add 900µl room temperature LB medium to the tubes. Incubate for 1 hour at 37℃ with shaking (~150rpm).

5) Plate 100µl of each transformation culture onto duplicate LB/ampicillin plates or LB/ampicillin/IPTG/X-Gal plates. If a higher number of colonies is desired, the cells may be pelleted by centrifugation at 1,000×g for 10 minutes, resuspended in 200µl of SOC medium, and 100µl plated on each of two plates.

6) Incubate the plates overnight (16~24 hours) at 37℃.

3. Identification of Recombinant Colonies by Colony PCR Method

1) Mixed reagents as described below into a 1.5 ml tube, divide this 250 μl mixture to 10 thin-wall tubes on ice. Every colony PCR reaction is 25μl. Adjust the amount of the mixture according to what you need.

          10×Taq DNA Polymerase Buffer                   25 μl

          dNTPs(10mmol/L)                                           5 μl

          T7 promoter primer (10μmol/L)                     5 μl

          T3 promoter primer (10μmol/L)                     5 μl

          Taq DNA Polymerase                                     10 Units

          ddH₂O                                                                up to 250 μl

2) Take cells from plates with sterile tips into 25μl mixtures.

3) Perform PCR cycles according to the following condition

94℃ for 5 minutes; 94℃ for 30 seconds, 42℃ for 30 seconds, 72℃ for 50 seconds, 30cycles.

4) PCR products from recombinant colonies are 660 bps, but these from negative ones are 160 bps (between T7 Promoter and T3 Promoter).

General Considerations

1. For 500bps insert DNA, > 80% of the whole transformants are recombinant colonies when molar ratios of insert DNA to the vectors are 1:1 to 100:1; but the most optimal molar ratios of insert DNA to the vectors are 2:1~10:1, at that time, >90% are recombinant ones.

2. Generally, the ratio of blue colonies is about 5~10% when LB/ampicillin/IPTG/X-Gal plates are used. Since the ratio of white colonies is > 90%, blue/white color selection is not needed.

3. When a ligation reaction with 30ng of pKRX-T (but without insert DNA) is set up as a background control, the number of colonies from the background control is about 10% of that from Control Insert DNA Cloning or PCR product cloning group.

4. The pKRX-T vector is from pBluescript II SK (+).

5. The control Insert DNA is gel-purified PCR product (500bps).

6. When identifying the recombinant colonies by PCR with T7/T3 promoter primers, pay attention that there is 160 bases between T7 Promoter and T3 Promoter.

7. Thaw pKRX-T vector and 10×T4 DNA Ligase Buffer at room temperature or 37℃.

Q & A

1. How to acquire more recombinant colonies?

1) The PCR product should be gel-purified to remove primer-dimers, multiple PCR products and other impurities.

2) Exposure to shortwave UV should be limited as much as possible because PCR product cannot be ligated due to pyrimidine dimers formed from UV overexposure.

3) Optimize the molar insert: vector ratios to 2:1~10:1.

4) Avoid multiple freeze-thaw cycles, otherwise pKRX-T vector and PCR product may lose 3´-T/A overhangs.

5) Ligation incubation must be longer than 3 hours. Optimal results are observed with an overnight ligation.

2.  How to calculate the appropriate amount of PCR product (insert) to include in the ligation reaction?

Use the following equation: [vector(ng) × size of insert(kb)÷size of vector(kb)] ×insert: vector molar ratio = insert(ng)

Example of insert: vector ratio calculation:

How much 0.5kb PCR product should be added to a ligation in which 30ng of 3.0kb vector will be used if a 5:1 insert: vector molar ratio is desired?

          30ng vector × 0.5 kb insert ÷3.0 kb × 5 = 25ng insert

3.  How to dilute T7 / T3 Promoter Primer to 10μmol/L?

Collect oligo DNA (1nmol) to the bottom of the tubes by centrifuging for a few seconds and add 100μl TE buffer to dilute the oligo DNA.