Jie:LabNotes/ASE/2008-2-29: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Jie deng
(New page: during the circularization reaction, the ratio of probe/target should be at least 125:1 or 250:1.<BR> for example: genomic DNA is 100ng/ul. How much CES22K probe(360, concentration is 9.3n...)
 
>Jie deng
No edit summary
Line 37: Line 37:
   tube 4: 95c 10min -> 60C overnight -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) -> 60C 1h -> 37C 1min <BR> -> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.
   tube 4: 95c 10min -> 60C overnight -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) -> 60C 1h -> 37C 1min <BR> -> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.


prepartion of SLN is needed calculated.
  prepartion of SLN is needed calculated.
  2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1uM dNTP
  2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.02uM dNTP
 
==PCR==
 
PCR reaction system
 
 
 
waiting for Alu 2 oligo
waiting for Alu 2 oligo

Revision as of 21:37, 27 February 2008

during the circularization reaction, the ratio of probe/target should be at least 125:1 or 250:1.
for example: genomic DNA is 100ng/ul. How much CES22K probe(360, concentration is 9.3ng/ul) is needed?

 since the molecular wight of one genome is 3pg, 200ng DNA is equal to 200ng/3pg.
the probe molecule needed is 200ng/3pg x 250. For each target and the total probe molecule is 200ng/3pg x 250 x 22,000.
the Mol of the probe is 200ng/3pg x 250 x 22,000 / (6x10 23) the concentration of probe is 9.3ng/ul. The length of each probe is approximately 90base (single strand oligoDNA). Since 1base = 330Da, the concentration of probe is 9.3ng/ul /(90x330) = 300nM the volume the probe needed is 400ng/3pg x 250 x 22,000 / {(6x10 23 阿佛加德罗常数)x70nM} = 2.0ul

Now add 1.5ul probe in reaction, the ratio of probe/DNA = 1.5x250/2 =188


 set the following circularization reactions:
 tube Standard 1: Standard reaction system
 tube Standard 2: Standard reaction system
 tube Standard 3: Standard reaction system
 tube 1: decrease dNTP concentration from  to 50uM to 1uM 
 tube 2: decrease dNTP concentration from  to 50uM to 0.02uM
 tube 3: change the annealing steps from 60C overnight to the mutiple steps
 tube 4: increase probe concentration from ratio 188:1 to 752:1
   
 reaction system                Stad tube 1   Stad tube 1   Stad tube 3    tube 1    tube 2     tube 3     tube 4
 H2O                              5.5ul         5.5ul          5.5ul       5.5ul      5.5ul     5.5ul        1ul 
 10x Ampligase Buffer               1ul           1ul            1ul         1ul        1ul       1ul        1ul
 probe                            1.5ul         1.5ul          1.5ul       1.5ul      1.5ul     1.5ul        6ul
 Jurkat Genomic DNA(100ng/ul)       2ul           2ul            2ul         2ul        2ul       2ul        2ul
 
 
 Reaction program: 
 
 Standard: 95c 10min -> 60C overnight -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) -> 60C 1h -> 37C 1min 
-> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold. tube 1: same as standard except that dNTP in SLN mix is 25uM (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1uM dNTP) tube 2: same as standard except that dNTP in SLN mix is 10uM (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.02uM dNTP) tube 3: 95c 10min -> 60C 1h -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) -> 60C 1h -> (95c 1min -> 60C 1h x 20cycles)
-> 37C 1min -> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold. tube 4: 95c 10min -> 60C overnight -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) -> 60C 1h -> 37C 1min
-> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.
 prepartion of SLN is needed calculated.
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1uM dNTP
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.02uM dNTP

PCR

PCR reaction system


waiting for Alu 2 oligo