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View additional product information for Superscript® Full Length cDNA Library Construction Kit - FAQs (A11181)
13 product FAQs found
This tends to occur more often when the clone contains a small insert, making it more likely that a hairpin will form between the similar attL1 and attL2 sites. To help with this, follow the suggestions below:
-Use a kit that will produce high-quality plasmid DNA such as the PureLink HiPure Plasmid Miniprep Kit.
-Use approximately 700 ng of DNA per sequencing reaction.
-Ensure that the clone has a valid insert by restriction enzyme digest with BsrG I. If the clone does not have an insert the restriction analysis will only show the vector backbone band of 2.4 kb. In such cases, the sequence of the empty clone is usually poor.
-Try a new set of sequencing primers.
-It is possible to use a blocking oligo as described in the following reference: Esposito D, Gillette WK, Hartley JL. (2003) Blocking oligonucleotides improve sequencing through inverted repeats.
Biotechniques 35:914-920.
-For particularly difficult clones, you may perform an LR transfer reaction of the clone into the destination vector of choice. attB sites are much smaller than attL sites, thus easier to sequence through.
Check to see if an insufficient amount of cDNA is being used in the BP recombination as this could lead to a low percentage of recombinants.
Low cDNA yield can result from a low RNA starting material. Use 0.5-5 µg starting mRNA for a standard library, check mRNA integrity and perform the reaction with the RNA control. Try radiolabeling protocols to assess efficiency of cDNA synthesis reaction. We would also suggest checking the transformation efficiency and Gateway enzyme efficiency.
Low cDNA yield can also result from the column not being prepared correctly for size fractionation. Let the columns drain completely before adding more buffer and ensure flow rate/drop size is correct.
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.
We would recommend using our SuperScript Full-Length cDNA Library Construction Kit
We recommend using our Platinum Taq DNA Polymerase High Fidelity enzyme for second-strand synthesis. We do not recommend a 2 min 95 degrees C activation step when using this enzyme. The antibody that inhibits Taq starts to dissociate at 60 degrees C. The standard primer extension protocol in the manual with incubations at 68 degrees C for 20 mins and 72 degrees C for 20 mins works well to extend 10 kB cDNA. We have not tested other enzymes for second-strand synthesis.
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 10e5-10e6 primary clones. We recommend using 5-10 µg starting mRNA with the SuperScript Full-Length cDNA Library Construction Kit.
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.
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.
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.
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.
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.
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