Easy cloning of all PCR products, including blunt and TA ends
This PCR Cloning Kit contains an optimized Cloning Mix containing a proprietary ligation enhancer and a linearized vector that uses a novel mechanism for background colony suppression to give a low background. It allows simple and quick cloning of any PCR amplicon, whether the amplification reactions are performed with proofreading DNA polymerases, such as Q5® which produce blunt ends; or nonproofreading DNA polymerases, such as Taq or Taq mixes (OneTaq®, LongAmp® Taq) which produce single base overhangs. This is possible due to “invisible” end polishing components in the master mix that are active during the ligation step only if needed. The kit also allows direct cloning from amplification reactions without purification, and works well whether or not the primers used in the PCR possess 5´-phosphate groups. To learn more about how the kit works, please view our video.
The following reagents are supplied with this product:
NEB # | Component Name | Component # | Stored at (°C) | Amount | Concentration |
E1202S | Multi-temperature |
Linearized pMiniT™ 2.0 Vector | N0312AVIAL | -20 | 1 x 0.02 ml | 25 µg/ml | |
Cloning Analysis Forward Primer | S1512AVIAL | -20 | 1 x 0.035 ml | 100 µM | |
Cloning Analysis Reverse Primer | S1513AVIAL | -20 | 1 x 0.035 ml | 100 µM | |
Amplicon Cloning Control | N0555AVIAL | -20 | 1 x 0.01 ml | 15 µg/ml | |
pUC19 Vector | N3041AVIAL | -20 | 1 x 0.025 ml | 50 pg/µl | |
Cloning Mix 1 | M1015AVIAL | -20 | 1 x 0.08 ml | 2.5 X | |
Cloning Mix 2 | M1016AVIAL | -20 | 1 x 0.02 ml | 10 X | |
NEB® 10-beta/Stable Outgrowth Medium | B9035SVIAL | 4 | 1 x 25 ml | ||
NEB® 10-beta Competent E. coli (Cloning Efficiency) | C3018AVIAL | -80 | 20 x 0.05 ml |
1. Use an insert:vector ratio of 3:1: A higher insert:vector ratio can actually result in fewer colonies. This is due to the fact that inserts may ligate to both ends of the vector, which will prevent the cloning of your amplicon insert.
2. Follow the protocol: The protocol has been highly optimized to have a low background; if you have inadvertently deviated from the optimized protocol (e.g., extended ligation incubation, overly-concentrated outgrowth), compensate by plating less outgrowth (< 50 µl) - Plating too much of the outgrowth can increase background, and cause problems with colony PCR. If you need more colonies, spread 50 µl of outgrowth onto each of multiple plates.
3. Important to stop ligations: If you wish to store your ligations to allow transformations at a later time, make sure your freezer is cold enough (- 20°C) to freeze the ligations. Or, you may quick freeze with a dry ice/alcohol bath before transferring the samples to -20°C. If you find your freezer-stored ligations have remained in liquid form, this may have allowed further low-level ligation of the vector backbone to occur. In this circumstance, plate less outgrowth (< 50 µl).
4. Do not incubate the transformation plates at room temperature. The slow growth rate of the cells at room temperature will increase the number of background colonies.
5. Add the cloning mixes 1 and 2 to the reaction last. Some people try to save time by preparing a mix of water, ligation master mix and pMiniT, aliquoting this to tubes and adding the insert DNA. This allows pMiniT to recircularize, since ligation can begin before the amplicon is added, and this may result in lower cloning efficiency.
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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
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