Noi/NOTES/2012-7-11

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Suggestions from lab meeting 2012.07.11[edit]

epMotion testing slide: http://genome-tech.ucsd.edu/LabNotes/index.php/noi:Lab_Presentation

Experiment set up 
~ 670ng of PCR product (very under estimated) amplified from Sergio's experiment (BSPP capture) in total 80ul was added to two columns on 96-well plate. Empty well with H2O was put between the well with DNA for contamination validation
 
File:2round bead layout.png

Elution volume1 = 30ul
Elution volume2 = 22ul
- Load 1ul of purified DNA on each well 
 File:ZhangLab 2 2012-07-10 18hr 26min bead2-1.jpg File:ZhangLab 2 2012-07-10 18hr 50min bead2-2.jpg
- Perform PAGE quantification to calculate recovery rate for each purification
1st round (to initial DNA amount): 80.2% STD 5.7%
2nd round (to 1st round): 78.7% STD 8.8%
2nd round (to initial DNA amount): 68.6% STD 7.7%
Test contamination by PCR
- 1ul of purified DNA (elution volume 22ul) was added to 50ul qPCR
 
Components	         1x   18xrxn mix
DNA template (10ng)	1.00	 0.00
100uM AmpFV6.3NH2	0.10	 1.80
100uM AmpRV6.3NH2	0.10	 1.80
2X KAPA MM             25.00   450.00
H2O	               23.80   428.40
Total	               50.00   900.00
===================================== 
aliquot 24ul , add DNA template 1ul

Program
98C 30sec --> (98C 10sec -> 58C 20sec-> 72C 20sec) x 35 cycles --> 72C 2min 

File:2012 07 10 epMotion Test Contamination.png
Things to be further optimized
EtOH dispension to the well (washing)
The tip position is very close to the well
Mixing with the lid on
Can not remove all EtOH from the well (~10ul). Have to remove manually 
Mixing 20% PEG requires pipetting
Discussion
Contamination sources?
Contamination between adjacent wells
Contamination from wells to reagents
How to improve?
Adjust aspiration and dispensing steps
Put lid on during shaking on thermomixer
Is plate/tube spinning required after mixing step
Is the contamination level acceptable (can not tell level of contamination)
Conclusion
1. AMPure bead purification by reusing the beads by epMotion give consistent results and high recovery rate 
2. Contamination were detected during AMPure bead purification by epMotion in 96-well plate
Comments from Dr. Zhang
1) Amounst of DNA should be varied in different amount to see the recovery rate comparing to high input I got this time, especially the low input DNA amount
2) To validate the sources of contamination, the reagents used in the testing like EB buffer should be added to the PCR