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cDNA Library Construction Kits

We would recommend using the CloneMiner™ cDNA Library Construction Kit (Cat. No. A11180) for construction of high-quality Gateway® cloning-compatible cDNA libraries without the use of restriction enzyme cloning. This system uses highly efficient recombinational cloning, resulting in a higher number of primary clones compared to standard cDNA library construction methods.

First-strand cDNA synthesis is most commonly primed using oligo(dT) or modifications of this sequence (such as primer-adapters) that bind to the poly(A) tail of mRNA. This priming method offers two major advantages: only poly(A)+ RNA is copied, and most cDNA clones begin at the 3’ terminus of the mRNA.

At the same time, oligo(dT) priming has certain limitations—some cDNA clones may not be full length (due to RNA secondary structure or pausing by reverse transcriptase) also, poly(A)- mRNA cannot be copied.

An alternative method is to use random hexamers, which, in theory, are capable of binding and priming throughout virtually any RNA template. Random hexamers, which may be used either by themselves or in combination with oligo(dT), have been instrumental in producing cDNAs containing more 5’ information than those primed with oligo(dT) alone. In addition, random hexamers can be used to generate cDNA libraries from poly(A)- mRNA and single-stranded viral RNAs.

Gateway® entry clone cDNA libraries are ready to transfer into suitable destination vectors for gene expression. You do not have to worry about restriction enzyme digestion of cDNA (which can decrease the insert size,) or vector prior to cloning.

The CloneMiner™ II kit offers the following improvements:

  • SuperScript® III RT instead of SuperScript® II RT
  • BP Clonase® II Enzyme Mix instead of BP Clonase® Enzyme Mix
  • Simplified cDNA fractionation protocol with no radioactive labeling of cDNA and 3 fractions collected

The kit includes a simple and robust protocol for the elimination of truncated mRNA. Magnetic beads coated with antibodies against the 5’ cap structure allows specific enrichment of 5’ capped–mRNAs (5’ capped–mRNA transcripts are by definition full length). Truncated mRNAs are thus eliminated from the library.

This is not recommended. pDONR™ 222 has the kan gene in the opposite orientation. This has been empirically noted to increase the transformation efficiency, which is needed to target the highest primary titer possible.

No, the pDONR™ 222 vector is not available as a standalone vector. However, it can be propagated in OneShot® ccdB Survival™ 2 T1R cells. After propagation, it is important to check that the ccdB gene has not been lost during propagation. Perform a comparative colony count between the propagated pDONR™ 222 transformed into ccdB Survival™ 2 T1R cells and TOP10 cells (or another sensitive strain). There should be 10,000x more cells when transformed into ccdB Survival™ 2 T1R cells.

We do not suggest using chemically competent cells. There may be as much as a 100-fold reduction in the number of primary clones if chemically competent cells are used.

Yes, we strongly recommend precipitating the DNA to avoid arcing with the electrocompetent cells following the BP Clonase® II reaction. The precipitated DNA can be resuspended in water instead of TE.

The CloneMiner™ II cDNA Library Construction Kit is recommended for nanoquantity libraries. A smaller amount of mRNA down to 50 ng can be used to construct nanoquantity libraries containing 105–106 primary clones. We recommend using 5–10 µg starting mRNA with the SuperScript® Full-Length cDNA Library Construction Kit.

The attB1 adapter in the SuperScript® Full-Length cDNA Library Construction Kit is double stranded because it increases efficiency of ligation to the single-stranded cDNA.

We recommend using our Platinum® Taq DNA Polymerase High Fidelity enzyme for second-strand synthesis. We do not recommend a 2 min 95°C activation step when using this enzyme. The antibody that inhibits Taq starts to dissociate at 60°C. The standard primer extension protocol in the manual with incubations at 68°C for 20 mins and 72°C for 20 mins works well to extend 10 kB cDNA. We have not tested other enzymes for second-strand synthesis.

The “pre-spin” is recommended as a precaution to ensure that the filter is tightly installed in the column. In some instances, users got less yield when the filter was not tight, and cDNA fragments passed through around the filter.

Gene Pool™ cDNA

Gene Pool™ cDNA was made using M-MLV reverse transcription at 42°C.

The key to generating full-length cDNA is to start with high-quality, intact RNA and to use the highest quality reagents in reverse transcription. All RNA used to synthesize Gene Pool™ cDNA is purified using pre-qualified reagents. After purification the RNA is treated with DNase to eliminate contaminating DNA which can lead to spurious bands when performing PCR. The cDNA is further analyzed for purity by UV spectrophotometry. To guarantee the quality of reverse transcription, each reaction is verified by amplifying fragments of the actin and clathrin housekeeping genes. The 5´ end of the 6 kb clathrin transcript must be successfully amplified for the cDNA to meet our strict quality standards. The ability to PCR amplify the 5´ end of the 6 kb gene demonstrates the integrity and processivity of the reverse transcription reaction which ensures full-length cDNA.

cDNA Libraries

Yes, the Gateway® LR reaction allows you to transfer a cDNA library made in Gateway® entry vectors into Gateway® destination vectors with no significant effect on the library's average insert size or insert size range. Thus, the library's complexity is maintained after the LR reaction. The library transfer reaction protocol is found in the CloneMiner™ II cDNA Library Construction Kit manual.

There is no recommended method that would maintain the complexity of the original library. The best option is to create a new library using the CloneMiner™ II cDNA Library Construction Kit. This kit will allow you to clone, via a BP reaction, all the generated cDNAs into a Gateway®-compatible vector.

It depends on whether the lambda vector has recombination (att) sites that are compatible with the design of our system. Without att sites, the library would definitely not be Gateway®-compatible.

Yes, but the pCMVSport vector does not have a eukaryotic origin of replication or selectable marker, so you can only screen for transient expression.

It depends upon the library. Only libraries in pCMVSport6 or pCMVSport6.1 have attB sequences.

The cDNA insert is cloned in the Not I/Sal I sites of the pCMVSport vectors. The Sal I site is the 5' end of the sense strand. Therefore, in order to make an in vitro transcribed/translated product from the sense strand, one would have to use the SP6 promoter.

Inserts cloned into lentiviral vectors should not have a poly(A) signal. The native poly(A) signal (AATAAA or something similar) will be amplified when using the oligo(dT) during cDNA synthesis. Thus, it will then become part of the cDNA library or its clones.

Since lentivirus is an RNA virus, during the synthesis of the RNA genome to be packaged, if there is a polyadenylation [poly(A)] signal in the insert, the RNA will be terminated prematurely. Almost any clone transferred from a Gateway® cDNA library will probably have a poly(A) signal, which, if inserted into a lentiviral vector, would end up terminating the viral RNA prematurely.

In order to circumvent premature termination of the lentiviral RNA, consider these recommendations:

The desired gene should first be isolated from the library, cloned into an entry vector such as pENTR™/D-TOPO® without the poly(A) signal (i.e., ATG to stop), and then transferred into the lentiviral vector.

If you are trying to establish a lentiviral expression library, you will probably have to go with a library that was amplified using random hexamers rather than an oligo(dT), since such a library would be less likely to include a poly(A) signal in the insert.

Size is not usually a problem. The insert size limit of lentivirus is ~5–6 kb (average insert size of the SuperScript® II Pre-made Libraries is ~1.5 kb).

Semisolid amplification of primary cDNA transformants minimizes representational biases that can occur during the expansion of plasmid cDNA libraries. We also recommend performing amplification at 30°C, which helps to stabilize unstable clones. See below for a detailed protocol.

CAUTION: Bottles of semisolid agar containing suspended colonies must be handled gently and incubated without disturbance. Rough handling or bumping of the incubator will cause microcolonies to fall out of solution. Incubators that have fans can cause colonies to fall out of solution.

Recommended Procedure:

  1. Prepare 2.0 L of 2X LB. Remove 200 mL of the 2X LB to make the 2X LB Glycerol (see below).
  2. Place a large stir bar and 1.35 g SeaPrep® (FMC) agarose into each of four 500 mL autoclavable bottles. Place bottles on stir plates. With the stir plate turned on, add 450 mL of 2X LB to each bottle (avoid the formation of large clumps of agarose).
  3. Autoclave these bottles of 2X LB agarose for 30 min.
  4. Cool bottles in 37°C water bath for ~2 hr until media reaches 37°C.
  5. After the media reaches 37°C, add the appropriate selection antibiotic. For ampicillin resistance markers, add carbenicillin to 50 µg/mL (preferred) OR ampicillin 200 µg/mL. Mix on stir plate. (Carbenicillin reduces satellite formation.)
  6. To each bottle, add 4 x 105–6 x 105 primary cDNA transformants (colonies from original library), and mix thoroughly on a stir plate for 2 min. Tighten caps.
  7. Place bottles in an ice water bath (0°C) such that the level of water in the bath is at the same level as the upper level of media in the bottle. Incubate 1 hr in the ice bath.
  8. Gently remove bottles from ice bath and wipe the water off the outside of the bottles. Loosen bottle caps. Place these bottles in a gravity flow incubator set at 30°C.
  9. Incubate these bottles for 40–45 hr without disturbance. Place a Sorvall GSA Rotor at room temperature the morning of harvest.
  10. Pour contents of bottles into Sorvall GSA bottles and centrifuge at 8,000 rpm for 20 min at room temperature (Caution: Make sure that the rotor was set at room temperature for at least two hours before adding the Sorvall GSA bottles. Rotors at 4°C will cause solidification of agar.)
  11. Pour off supernatant.
  12. Resuspend the cells in a total volume of 100 mL 2X LB Glycerol (12.5%). Remove two 100 µL aliquots for plating, further analysis, and colony estimate. Cells can be filtered through sterile cheesecloth to remove agarose clumps if present.
  13. Subdivide the cells into ~10 mL aliquots (Note: It is useful to make a number of 1 mL and 100 µL aliquots.) and place at -70°C. Store at -70°C.

Frozen cells can then further amplified in liquid at 30°C to obtain DNA. Use 2.5 x 109 cells per 100 mL growth medium for further expansion of library.

2 X LB Recipe:
20 g Tryptone
10 g Yeast Extract
10 g NaCl
Bring to 1,000 mL with H2O.

2 X LB Glycerol (12.5%) Recipe:
175 mL 2 X LB
25 mL Glycerol (100%)
Filter sterilize and store for up to two months at room temperature.

Reference:
Kriegler M (1990) Gene Transfer and Expression: A Laboratory Manual. Stockton Press, New York, NY.
Hanahan D, Jessee J, and Bloom FR (1991) Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol 204:63-113.

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