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Frequently Asked Questions

Cloning

1. Should I use the high copy pSMART-HCKan and -HCAmp vectors or the low copy pSMART-LCKan and -LCAmp vectors?
The high-copy vectors are used for most cloning and library construction projects. They have been shown to maintain large inserts (> 10kb), inserts with strong promoters, or toxic coding sequences. However, inserts with high AT content (>60-65%) may be unstable in the high copy number vector.

The low copy kits are best if the target DNA is suspected to be unstable or difficult to clone. Libraries from several microbial genomes have been shown to be stable in the low copy vector but unstable in the high-copy vector.

Inserts appear to be more stable in the kanamycin-resistant versions of the vectors, for reasons that are not well defined at this time.

2. What is the copy number and plasmid yield of the vectors?
The copy number of pSMART-HC is similar to pUC19, about 300-500 copies per cell. It yields ~ 20-40 ug of plasmid per 1 ml culture. The copy number of pSMART-LC is similar to pBR322, about 15-30 copies per cell. It yields ~ 0.5 ug of plasmid per 1 ml culture.

3. Can I clone PCR products with the CloneSmart Kits?
Yes, but the PCR product must be treated with the PCRTerminator® kit to generate ends that are compatible with the blunt insertion site of the CloneSmart vectors. Lucigen's PCR-SMART Kits are another option.

4. Can I store the completed ligation reactions and transformed cells?
Yes. After the ligation reaction has been denatured at 70°C for 15 minutes, it can be stored at -20° C indefinitely.The transformed cells that are left over after plating can be stored by adding sterile glycerol to a final concentration of 15%. Store the cells at -80°C.

5. Can other electroporation competent or chemically competent E. coli strains be used for transformation?
Yes, the CloneSmart ligations can be transformed into other strains of E. coli, but fewer clones will likely be obtained. Lucigen's E. cloni electrocompetent cells yield over 20 billion clones per microgram of pUC19 DNA. Using less efficient cells will result in a proportionate decrease in the number of colonies obtained. To obtain the maximum number of transformants, use E. Cloni 10G SUPREME cells, which produce approximately >4×1010 cfu/µg.

6. Does the ligation reaction need to be extracted or precipitated before transformation?
No. One microliter of the ligation mix can be used directly to transform electrocompetent cells.

7. What are the sequences of the primers?

SL1 is: 5'-CAG TCC AGT TAC GCT GGA GTC
SR2 is: 5'-GGT CAG GTA TGA TTT AA A TGG TCA GT

The pEZSeq primers are identical to the M13 Forward and Reverse Primers:
Z-Rev (M13 Reverse, -48): 5'-AGC GGA TAA CAA TTT CAC ACA GGA
Z-For (M13 Forward, -41): 5'-CGC CAG GGT TTT CCC AGT CAC GAC

These are also listed in the manual for each kit.

8. What are the sequences of the vectors?
The vector sequences can be obtained from GenBank. The Accession numbers are as follows:

pSMART-HCKan: AF532107
pSMART-LCKan: AF532106
pSMART-HCAmp: AF399742
pSMART-LCAmp: AY090111
pEZSeq-Kan: AF532108
pEZSeq-Amp: AF532109

9. How do I screen pSMART clones for inserts?
In a typical ligation/transformation, there is no need to screen for inserts, since over 99% of the clones will have inserts. However, it is possible to use PCR with the SL1 and SR2 primers to confirm the presence or size of the inserts. PCR can be performed on the transformed colonies or on DNA prepared from the colonies. In addition, the cloning site of the pSMART vectors is flanked on both sides by EcoRI and EcoRV restriction sites, so a digest with either enzyme will excise the insert from the vector for gel analysis.

10. I have a very high background of empty vector. What went wrong?
The major cause of empty vector transformants is contamination of the insert DNA either with kinase or exonuclease. Exposing the CloneSmart vectors to either of these enzymes may allow the vector to self-ligate. Be sure that the DNA to be used for cloning has been purified by gel electrophoresis, spin column chromatography, or extraction before adding it to the ligation reaction.

In addition, DNA samples isolated from agarose gels are easily contaminated with low-molecular weight fragments, often less than 100 bp. These micro-fragments are cloned with VERY high efficiency, and the resulting clones appear to be empty. Therefore, it is very useful to sequence several clones that appear to be empty vector to determine whether there are small deletions of the vector or small fragments cloned into the vector.

11. I am getting very few clones from my transformations. What is wrong?
There are several possible reasons for low numbers of clones. The most common reasons are too little DNA or poor quality DNA used for ligation. Be sure to quantitate the DNA carefully and check its integrity by agarose gel electrophoresis.

Another problem may be that the ends are not blunt or not phosphorylated. Lucigen's DNATerminator® Kit is an extremely efficient method for repairing the ends of DNA fragments generated by physical shearing or restriction digestion. Standard methods of end repair involving T4 DNA polymerase or mung bean nuclease may be used instead, but the cloning efficiency is likely to be lower.

A potential problem with cloning genomic DNA is that it may have modifications that are not tolerated by E. coli cells. Try using the low-copy number CloneSmart Kit for improved results. See the CloneSmart Manual for additional information (Appendix E: Troubleshooting).

12. Why are my sequence reads low quality (low signal, high background or "noise")?
Poor sequence reads are often due to low quality sequencing primers or low quality or quantity of plasmid DNA. Test the quality of your primers by comparing their performance with the control primers provided with the CloneSmart Kits. Confirm the integrity and quantity of your DNA samples by gel electrophoresis with size and mass standards.

Lucigen has developed novel transcription-free cloning systems designed to improve cloning yields, reduce cloning bias and improve sequencing throughput.

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