August Library (8/24/09)

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August Library[edit]

Probes, Samples, and Calculations[edit]

Probes:

Jan09-1:  12.4 ng/ul,  46417 probes;  376 nM
Jan09-2:  16.3 ng/ul,  53493 probes;  494 nM
Jan09-3:  7.8 ng/ul,   53828 probes;  236 nM

Samples:

Sample #	        Sample name	       Amount	     Concentration
  1	            MH001 1+2 xHeaIII	       10 ul	      102.7 ng/ul
  2	                 MH010B xMboI	        6 ul	      183.4 ng/ul
  3	                 MH010B xDpnI	       10 ul  	      100 ng/ul
  4	                   TCF #3	        6 ul	      200 ng/ul
  5                        DK4 #2	        17 ul	      58.9 ng/ul
  6	                   TK4 #2	        18 ul	      55.9 ng/ul
  7	                   TK9	                12 ul	      89.4 ng/ul 
  8	                   Pol II	        10 ul	      99.7 ng/ul

Calculations

Probe: Target ratio is 100:1
The mass of one genome is 3pg: 200ng of DNA is equal to 200ng/3pg.
The amount of probe needed is 200ng/3pg * 250 for each target, and the total amount of probe is 
(200ng/3pg * 100 * 46417)/(6.0 * 10^23)

Example
Assume the concentration of probe Jan09-1 to be 10ng/ul. Knowing the length of the probe is ~100 bases and 1 base = 330 Daltons (1bp = 2 bases = 660 Da).
Solve: Concentration of Jan09-1 probe: 10ng/ul / 33000 = 303 nM
    	Volume of probe needed= (200ng/3pg * 100 * 46417)/(6.0 * 10^23 * 303 nM) = 1.73ul
Volumes needed of each sample:
Sample 1 – 200ng / 102.7ng/ul =  1.947ul
Sample 2 – 200ng / 183.4ng/ul =  1.091ul
Sample 3 – 200ng/ 100.0ng/ul =   2.000ul
Sample 4 – 200ng/ 200.0ng/ul =   1.000ul
Sample 5 – 200ng/ 58.9ng/ul =    3.396ul
Sample 6 – 200ng/ 55.9ng/ul =    3.578ul
Sample 7 – 200ng/ 89.4ng/ul =    2.237ul
Sample 8 – 200ng/ 99.7ng/ul =    2.006ul

Reaction Setup[edit]

Sample Number:                1            2       3       4       5       6      7       8
Sample DNA	            1.95ul      1.09ul	2.00ul	1.00ul	3.40ul	3.58ul	2.24ul	2.00ul
10x AmpLigase Buffer	    2.00ul	2.00ul	2.00ul	2.00ul	2.00ul	2.00ul	2.00ul	2.00ul
Jan09-1	                    1.40ul	1.40ul	1.40ul	1.40ul	1.40ul	1.40ul	1.40ul	1.40ul
Jan09-2	                    1.20ul	1.20ul	1.20ul	1.20ul	1.20ul	1.20ul	1.20ul	1.20ul
Jan09-3	                    2.50ul	2.50ul	2.50ul	2.50ul	2.50ul	2.50ul	2.50ul	2.50ul
ddH2O	                    10.85ul	11.81ul	10.90ul	11.90ul	9.50ul	9.32ul	10.66ul	10.90ul
                                                 Total Volume/Reaction = 20ul
94C, 2min -> -0.2C/sec to 60C -> 60C, 24h -> add 1ul SLN mix (dNTP 0.5mM, Stoffel 2U/ul, AmpLigase 4U/ul) -> 60C, 24h -> 94C, 1min -> 37C, 1min -> add 2ul Exo I&III -> 37C, 2h --   
90C, 5min -> 4C hold

PCR

(per reaction)
Template				    10ul
iProof Master Mix	    	            50ul
100uM AmpF6.3Sol		           0.2ul
100uM AmpR6.3Sol		           0.2ul
50x SYBG I			       	   0.2ul
ddH2O				            40ul
98C, 30sec -> (98C, 10sec -> 60C, 20sec -> 72C, 15sec) x 5 -> (98C, 10sec -> 72C, 20sec) x 20 - > 72C, 3min -> 4C hold