Noi/NOTES/2012-5-14

From ZhangLabWiki
Jump to navigation Jump to search

Probe production for the HiResChrPaint project[edit]

Production PCR[edit]

  • V4 pool for all four sets (12 reaction for each set)
Components Volume (ul) 49x rxn mix
20nM oligo pool 0.20 0.00
2x Kapa SYBG qPCR MM 50.00 2,450.00
100uM pAP1V41U 0.40 19.60
100uM AP2V4 0.40 19.60
2mM aa-dUTP 3.00 147.00
H2O 46.00 2,254.00
Total volume (ul) 100.00 4,900.00

- Aliquot 1197.6 ul in 1.5 mL tube, add 2.4ul of each oligo pool to each tube -> aliquot 100ul of reaction mix into strip tube

Program 
95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold.
#1-V4 -> 16 cycles #2-V4 -> 16 cycles #3-V4 -> 14 cycles #4-V4 -> 15 cycles (Note: take the average of the qPCR result) File:2012 05 14 Probe-V4-pool.png
  • V6 pool for all four sets (12 reaction for each set)
Components Volume (ul) 49x rxn mix
20nM oligo pool 0.20 0.00
2x Kapa SYBG qPCR MM 50.00 2,450.00
100uM pAP1V61U 0.40 19.60
100uM AP2V6 0.40 19.60
2mM aa-dUTP 3.00 147.00
H2O 46.00 2,254.00
Total volume (ul) 100.00 4,900.00

- Aliquot 1197.6 ul in 1.5 mL tube, add 2.4ul of each oligo pool to each tube -> aliquot 100ul of reaction mix into strip tube

Program
95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 15 -> 72C 2min -> 15C hold.
#1-V6 -> 15 cycles #2-V6 -> 14 cycles #3-V6 -> 14 cycles #4-V6 -> 14 cycles (Note: take the average of the qPCR result) File:2012 05 14 Probe-V6-pool.png - Ethanol precipitation. - Pool amplified amplicons (total volume 1,200ul each set) into 15mL tube - Add 4ul of Glycoblue - Add 120ul of 3M NaoAc pH5.5 - Add 3mL of 100% EtOH - Put the tubes at -80C for 1hr - Centrifuge at 3,000rmp for 30min, 4C - Remove spnt. add 600ul of 75% EtOH and transfer the DNA pellet to 1.5 mL tube - Spin at 14,000rmp for 5min - Dry the pellet in the hood and resuspend with 100ul H2O - Re-purified each amplicon with two Qiaquick columns, eluted with 40ul EB => total volume 120ul each. - Measure concentration with Nanodrop. Probe IDs Conc. (ng/ul) Yields in 120ul (ug) #1-V4 83.70 10.04 #2-V4 125.10 15.01 #3-V4 110.10 13.21 #4-V4 133.80 16.06 #1-V6 122.50 14.70 #2-V6 106.20 12.74 #3-V6 132.20 15.86 #4-V6 101.70 12.20

DpnII digestion[edit]

  • Note: Since I ran out of DpnII and the new tube I ordered hasn't come in yet. I have the old tube already expired 07/2011 ~20ul, but still works and the old tube from Dinh (expired 07/09 -> not sure about activity). To test if enzyme still works well then I set up digestion reaction in 50ul and also vary DNA amount between 2.5ug and 4ug to see if 4ul of enzyme can digest 4ug of DNA in 50ul completely or not.

This just to optimize reaction, so I set up digestion reaction in small volume.

Components	        Volume (ul)	          
DNA #1-V4(2.5 ug)	 29.50 => equivalent to 5ug DNA in 100ul reaction	      
10X DpnII Buffer	 5.00	
DpnII	                 4.00 (10U/ul => 40U)	
H2O	                11.50	
Total	                50.00	
  
Components	        Volume (ul)
DNA #4-V4(4 ug)         29.50 => equivalent to 8ug DNA in 100ul reaction
10X DpnII Buffer	 5.00
DpnII	                 4.00  (10U/ul => 40U)
H2O	                11.50
Total	                50.00

File:ZhangLab 2 2012-05-14 19hr 20min DpnII-digestion-30min.jpg File:ZhangLab 2 2012-05-14 19hr 20min DpnII-digestion-1hr.jpg

  • From the result above, there is no different between DpnII expired in 07/09 and 07/11. Even prolong incubation for 1 hr, didn't increase the digestion of DNA.
  • I think it's possible that
    • Too much DNA in the reaction (but I used to digest 6-7ug with DpnII in 100ul reaction for DMR 330k probe production?)
    • Or the enzyme activity is not fully active?.
  • However, I need to get completely digest the DNA in these tubes by adding DpnII (add 50ul of 1x DpnII buffer containing 4ul more of Dpn II (40units). For the rest of DNA, I will wait for the new tube of enzyme and optimize DNA digestion again (DNA amount, enzyme units, and incubation time) since I'm not sure that incomplete digestion I observed this time because of enzyme activity or DNA amount.
Info. from neb.com
  Unit definition of DpnII
  "One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA (dam-) in 1 hour at 37°C in a total reaction volume of 50 µl."
  Size of Lambda DNA ~48,502bp --> how many DpnII digestion sites, roughly check > 100 sites
  Size of oligos in this project ~100bp --> 1 DpnII digestion site (~485x size difference or 485 molar ratio with the same amount of DNA)
  Let say if we need about 5x units of DpnII in digest lambda DNA to digest the 100bp oligos with the same amount. This means 1ug of oligo requires 5U of DpnII, but normally we add more enzyme than the unit definition ~3-5x, so may add 15-25 (1.5 -2.5ul, 10U/ul) units to digest 1ug of oligos.
  I might try these conditions
    - 5ug DNA in volume 100ul, DpnII(3X)= 5ug * 5x of amount used for lambda DNA * 3x excess amount = 75 units or 7.5ul
    - 5ug DNA in volume 100ul, DpnII(4X)= 5ug * 5x of amount used for lambda DNA * 4x excess amount = 100 units or 10ul 
    - 5ug DNA in volume 100ul, DpnII(5X)= 5ug * 5x of amount used for lambda DNA * 5x excess amount = 125 units or 12.5ul too much? final % of glyceral => 12.5ul *50%glyceral in 100ul => >5% glycerol v/v
    Might set up small reaction 20ul
 Components	        Condition1   Condition2	   Condition3
 DNA (1ug), #2-V4	8.00	         8.00	      8.00
 10X DpnII buffer	2.00	         2.00	      2.00
 DpnII	                1.50	         2.00	      2.50
 H2O	                8.50	         8.00	      7.50
 Total 	       20.00	        20.00	     20.00
- Sampling ~0.5ul DNA from the reaction (50ng) at 30min, 1hr,  and 2 hr of digestion and check in 6% TBE gel. This optimization may spend much time, but it is necessary if we need completely digestion and no PAGE size-selection.
File:ZhangLab 2 2012-05-15 14hr 17min TestdpnII-1.jpg
1: condition1 
2: condition2
3: condition 3
From the image above, the digestion is still incomplete and there is no different between using different amount of enzyme or or incubation time. I think this is unlikely about enzyme activity. The last two lanes are the reaction from yesterday that I added more fresh enzyme (double volume) in 1X DpnII buffer. However I didn't see the different from the digestion from yesterday as the image shown above. From my experience when I digest the DMR 330k probe, I alway got completely digest by this enzyme. I then check the sequence of reverse primer AP2V6 and AP2V4 to see the present of DpnII reognition site.
  • Additional reverse primer info.
    • AP2V6: 5’-/5Phos/CACGGGTAGTGTGTATCCTG-3 and ATC in the oligo backbone
    • AP2V4: 5’-/Phos/TAGCCTCATGCGTATCCGAT-3’ and C in the oligo backbone (I guess)
    • DpnII recognition site: top strand --> 5'-...|GATC... 3', bottom strand 3'-...CTAG|...-5'
  • There is nothing wrong with the primer sequences, and I'm just realized that in this reaction some T will be replaced with aa dUTP. This is possible that the T residue in the DpnII recognition site is randomly replaced with aa dUTP and this make the digestion incomplete even I vary different amount of enzyme and incubation time.
  • Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-16