Sediment in the substrate tube in the XLR70

[mohdsos]

Hi everyone,

I've had an issue with one previous run. I've received a normalized and amplified RNA library.
I've titrate the library and ended up with:

cpb enrichment
0.5 1.12%
2 2.79%
4 3.38%
8 4.01%

I've scaled up the cpb after blotting the graph on excel, and calculated the cpb
to be to be 25-30 cpb. I am trying to achieve ~12% to get 4,000,000 beads to deposit it on 1 full PTP. however there was a lot of dot mixed wells and around 80% failed beads.

GACT (Library) Region
1 2 Total
Raw Wells 740,389 741,298 1,481,687
Key Pass Wells 706,227 700,576 1,406,803
Passed Filter Wells 83,862 82,674 166,536
Total Bases 11,280,758 11,271,684 22,552,442

Length Average 134.52 136.34 135.42
Length Std Deviation 80.56 81.24
Longest Reads Length 744 918 918
Shortest Reads Length 40 40 40
Median Reads Length 110.0 113.0 111.0


any suggestions ?

I don't know if the FLX reached the 200 cycles. or less

[pmiguel]

Did you denature your library before adding it to emPCR?

What was your % enrichment on the full PTP run you give results for?

What cDNA library construction method was use?

--
Phillip

[mohdsos]

yes I did.
13% , but I deposited 2M, yet in the wells are ~700,000
I've just received the lib , I didn't prepare it.

[pmiguel]

Did you run an Agilent chip on the library for QC? If so how did that look?

--
Phillip