No. The strand displacing amplification tolerates impurities in crude lysates very well.
Shipped on gel ice, needs to be kept at -20 °C or lower on receiving.
Yes. As long as they recognize:
Also, most of the third party kits configure primers at 10 uM, our protocol requires 25 uM, remember to reduce the final SPRI beads elution volume from 10 uL to 7 uL to make room for the primers.
>1,000 bp. Using DNA ladders that include fragments from 100 bp to 50,000bp, we found only when > 1,000 did the fragments get represented in the sequencing library generated by AFT™ Library Prep kit.
The possible reasons for no library amplification could be:
1). If single cell is sampled by dilution, the concentration of bulk cell suspension might be overestimated. Also by Poisson distribution, more than half of the sampling would contain no cell at all. Try to make three aliquots and make library from each of them.
2). If the single cell is collected by cell sorting, the tube may not catch the cell, try to collect three or more single cell in as many tubes and make library from each of the collection.
3)The DNA might be degraded and reduced to less than 1 kb; AFT ™ technology prefers to work on DNA longer than 1 kb. Try to keep cells intact before the procedure.
4). Excessive pipetting and vortexing during the workflow might reduce the target DNA to shorter than 1 kb, avoid excessive pipetting or vortexing.
5) When recovering the elution from SPRI beads, leave at least 3 uL behind, don’t try to recover all.
6). The third-party index primers are not compatible; some index primers are based on Nextera transposase recognition sites, not the standard Illumina Read1 and Read2 sequences. The correct index primer structures should be like below: