Hosuk:LabNotesMDA/2013-6-17
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MDA with PGP1F, 3th, Result[edit]
- Sequencing result was not good! (Kun's Labnote @06/15)
- Much worse than previous results.
Comments[edit]
- There are several issue with this batch of data: First, based on the SeqMonk coverage plot, DNA amplification is extremely sparse, and the cross-talk among different reactors is stronger. The latter could be also due to the read mapping.
- Second, there isn't clear evidence of big DNA molecules separated into individual reactors.
- Third, the clonal rate is much higher, which is probably also due to the poor amplification.
- Overall, there is something wrong with this experiment, either due to slow/incomplete mixing, or low KOH concentration.
- The next experiment needs to be design carefully to distinguish these two.
Next Plan[edit]
Minimizing mixer time[edit]
- Since one valving moves roughly 1/20 of entire volume of mixer, 20 valving will make 1 turn, and 10 turns (200 valving) would be enough for mixing (even distribution of DNAs in ring mixer). If 1 sec period for valving (because of slow flow speed), 200 sec = about 4min could be enough for complete mixing. Previous time I ran mixer for 10min, but if I try shorter mixing time (4 ~ 5min), then I could reduce remaining of DNAs
Separate pressure source for mixer valves from other valves[edit]
- By decreasing mixer valve pressure less than other valve pressure, I can minimize the area of the the membrane of mixer valves touches to the bottom of mixer channel. This could be reduce DNA damages from valving as well as remaining of DNAs under the valve area.
- I've already separate pressure source line of mixer valves from other valves, so I could find proper pressure with one or two test.
Revision of device design[edit]
- I could change ring mixer part if current design doesn't work. One idea is using vertical chamber for denaturing without using ring mixer. One or two valves are placed on the chamber. Even if the valves are not closed completely, they could distribute DNAs by creating turbulence. This would allow less fragmented DNAs and less remaining while transferred to compartments.
- But this design change is lower priority.
For the next experiment, (considering KOH concentration)[edit]
- Same 133mM KOH concentration,but 5min mixing, 1sec valving period, reducing valving pressure
- 250mM KOH with 5min mixing, 1sec valving period, reducing valving pressure
- I'll estimate amount of amplicon by qPCR after MDA reaction.