Products
Title: Can NadPrep NanoBlockers be diluted for use? What is the maximum total library input that can be blocked?
No. They can effectively block a total library input of up to 6 µg during hybridization capture when starting with 500 ng/library and performing 12 multiplexing cycles.
Title: Low library yield?
| Possible Reason | Solution |
|
Inaccurate quantification of input DNA a. Low input DNA with BS module. b. Compared with the EM module, the BS module needs 3 additional PCR cycles to reach the similar recovery efficiency. |
The Qubit fluorometer is recommended for the quantification of fragmented DNA. Increase PCR cycles; Convert low input DNA with EM module. |
Title: Low conversion efficiency (<99%) of unmethylated cytosine ?
| Possible Reason | Solution |
|
Contamination of post-converted library a. The BS module is out of date. b. The oxidation-caused crystallization of bisulfite may significantly inhibit the conversion efficiency. Input DNA is too much. |
a. Use filter tips. b. Replace the gloves and change experiment table after the conversion process. a. Use the BS module within the period of validity. b. Minimize the exposure to air. For EM module, the input DNA should be under 200 ng. |
Title: What is the possible reason for the less library yield or longer size distribution of library than expected after digestion？How to solve it ?
|
Possible Reason |
Solution |
|
The DNA sample contains more than 1 mM of EDTA |
Purify the DNA by using magnetic beads or spin columns,and dissolve the purified DNA with TE Solution. |
|
The DNA sample contains impurities |
High quality DNA input, with OD260 /OD280 =1.8-2.0 and OD260 /OD230 =2.0-2.5. The residual guanidine salt and proteins may suppress the fragmentase activity. Purify the DNA by using magnetic beads or spin columns,and dissolve the purified DNA with TE Solution. |
|
Fail to thoroughly mix the reaction mix |
Mix thoroughly and centrifuge to collect the contents to the bottom of the tube before initiate the reaction. |
|
Fragments self-ligation |
Thoroughly read the manual before operation. |
Title: What is the possible reason for the shorter size distribution of library than expected after digestion? How to solve it ?
|
Possible Reason |
Solution |
|
The DNA is seriously degraded |
Degraded DNA, such as FFPE C DNA, may result in short fragments (150-200 bp) without change of the same fragmentation condition. |
|
The fragmentation process lasts too long |
Avoid operating multiple samples at one time. |
|
The operation under room temperature lasts too long |
Ensure all the processes operated on ice beforeloading into the thermo cycler. |
Title: How to explain the appearance of the adapter peak in size distribution?
Possible Reason：Inappropriate dilution of adapters
Solution：For low DNA input, adapters should be diluted as recommended prior to use.
