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cDNA Library Construction
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.
A low average insert size can occur due to contamination with attB1 adapter, mRNA sample partially degraded, a problem with size fractionation, and/or not enough starting mRNA.
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.
<p>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:</p>
<li>Use a kit that will produce high-quality plasmid DNA such as the PureLink® HiPure Plasmid Miniprep Kit.</li>
<li>Use approximately 700 ng of DNA per sequencing reaction.</li>
<li>Ensure that the clone has a valid insert by restriction enzyme digest with <i>Bsr</i>G 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.</li>
<li>Try a new set of sequencing primers.</li>
<li>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.<br><i>Biotechniques</i> 35:914-920.</li>
<li>For particularly difficult clones, you may perform an LR transfer reaction of the clone into the destination vector of choice. <i>att</i>B sites are much smaller than <i>att</i>L sites, thus easier to sequence through.</li>
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