August Library (8/24/09): Difference between revisions

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==Probes, Samples, and Calculations==
=August Library=
 
===Probes, Samples, and Calculations===




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  Volumes needed of each sample:
  Volumes needed of each sample:
  Sample 1 – 200ng / 102.7ng/ul = 1.947ul
  Sample 1 – 200ng / 102.7ng/ul = 1.947ul
  Sample 2 – 200ng / 183.4ng/ul = 1.091ul
  Sample 2 – 200ng / 183.4ng/ul = 1.091ul
  Sample 3 – 200ng/ 100.0ng/ul = 2.000ul
  Sample 3 – 200ng/ 100.0ng/ul =   2.000ul
  Sample 4 – 200ng/ 200.0ng/ul = 1.000ul
  Sample 4 – 200ng/ 200.0ng/ul =   1.000ul
  Sample 5 – 200ng/ 58.9ng/ul =    3.396ul
  Sample 5 – 200ng/ 58.9ng/ul =    3.396ul
  Sample 6 – 200ng/ 55.9ng/ul =    3.578ul
  Sample 6 – 200ng/ 55.9ng/ul =    3.578ul
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  Sample 8 – 200ng/ 99.7ng/ul =    2.006ul
  Sample 8 – 200ng/ 99.7ng/ul =    2.006ul


==Reaction Setup==
===Reaction Setup===
  Sample Number:                1            2      3      4      5      6      7      8
  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
  Sample DNA             1.95ul      1.09ul 2.00ul 1.00ul 3.40ul 3.58ul 2.24ul 2.00ul
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  90C, 5min -> 4C hold
  90C, 5min -> 4C hold


PCR
''' PCR'''
 
  (per reaction)
  (per reaction)
  Template     10ul
  Template     10ul
  iProof Master Mix                 50ul
  iProof Master Mix                 50ul
  100uM AmpF6.3Sol           0.2ul
  100uM AmpF6.3Sol           0.2ul
  100uM AmpR6.3Sol           0.2ul
  100uM AmpR6.3Sol           0.2ul

Latest revision as of 04:23, 14 September 2009

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