Jie:LabNotes/ASE/2008-2-29

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Optimization of the padlock captureing efficiency

Design the optimization of the padlock capturing efficiency

 1. set three standard tubes. 
   S1 is the standard system as control for the whole experiment.
   S2 use the AluI to cut the circle DNA after Lambda exonuclease incubation, try to increase the efficiency of PCR.
   S3 use the ddH2O as the control of AluI.
 2. set four optimization tubes
   Tube 1 decrease the dNTP concentration in the SLE mix, try to decrease the possible activity of   to cut the circle.
   Tube 2 decrease the dNTP concentration with cascade in the SLE mix.
   Tube 3 change the annealing step from 60C overnight to several steps to increase the chance of correct annealing
among probes and targets. Tube 4 increase the probe concentration to increase the efficiency of annealing.

during the circularization reaction, the ratio of probe/target should be at least 125:1 or 250:1.
for example: genomic DNA is 200ng/ul. How much CES22K probe(390, 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 200ng/3pg x 250 x 22,000 / {(6x10 23 阿佛加德罗常数)x300nM} = 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, Alu I incubation before PCR
 tube Standard 3: Standard reaction system, control for Alu I incubation before PCR 
 tube 1: decrease dNTP concentration from  to 0.2uM to 4nM (This probe set is SNP probes, so the dNTP as Standard
is 0.2uM instead of 50uM for exon probes) tube 2: decrease dNTP concentration from to 0.2uM to 0.08nM 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 (CES22K,#390)              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 1: 95c 10min -> 60C overnight -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2uM dNTP) -> 60C 1h
-> 37C 1min -> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.
 Standard 2: 95c 10min -> 60C overnight -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2uM dNTP) -> 60C 1h
-> 37C 1min -> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 37C 1min -> add 0.5ul ALuI guide oligo
-> denaturing at 94C 3min -> 37C 1min -> add AluI(10U/ul) 1ul -> 37C 1h -> 75C 10min -> 4C hold.
 Standard 3: 95c 10min -> 60C overnight -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2uM dNTP) -> 60C 1h
-> 37C 1min -> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 37C 1min -> add 0.5ul ddH2O
-> denaturing at 94C 3min -> 37C 1min -> 37C 1h -> 75C 10min -> 4C hold.
 tube 1: same as standard 1 except that dNTP in SLN mix is 4nM (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 4nM dNTP) 
 tube 2: same as standard 1 except that dNTP in SLN mix is 0.08nM (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.08nM dNTP) 
 tube 3: 95c 10min -> 60C 1h -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2uM 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; 0.2uM dNTP) -> 60C 1h
-> 37C 1min -> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.
 prepartion of SLN 1ul:
 Storage reagents: AmpliTaq Stoffel fragment(10U/ul), AmpLigase(5U/ul),dNTP(10mM)
 Diltue dNTP concentration from 10mM to 1uM: 1ul dNTP(10mM) is added into 1ml ddH2O to get 10uM 
 *add 1ul dNTP(10uM) to 19ul ddH2O to get 0.5uM dNTP
 *add 1ul dNTP(10uM) to 1ml ddH2O to get 10nM dNTP 
 *add 1ul dNTP(10nM) to 49ul ddH2O to get 0.2nM dNTP

                                                                     ddH2O     Ligase Buffer   Ligase     Amplitaq    dNTP      
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2uM dNTP:     0.2ul         0.1ul       0.1ul       0.2ul    (0.5uM)0.4ul
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 4nM dNTP:       0.2ul         0.1ul       0.1ul       0.2ul    (10nM)0.4ul     
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.08nM dNTP     0.2ul         0.1ul       0.1ul       0.2ul    (0.2nM)0.4ul
 
 Preparation of AluI Oligo:
 RE-AluI-v6: ATCTGCAGCTGAATA  Tm=43C MW=4,576  4.0OD=27nmol=0.12mg
 Dilute with 270ul ddH2O to get the concentration of 100nM

PCR

PCR reaction system

 reaction system                                                         x14
 H2O                                                32.8ul            459.2ul                   
 10x JumpStartTaq Buffer                               5ul               70ul                   
 MgCl2(25mM)                                           3ul               42ul 
 dNTP(10mM)                                            1ul               14ul                  
 AmpSolV6F2/R2 mix(10uM)                               2ul               28ul                 
 50x SYBG I                                          0.2ul              2.8ul                   
 JumpStartTaq                                          1ul               14ul                   
 Products of Circluarization                           5ul               70ul                   
 Total                                                50ul              700ul

Run the program on Opticon4 realtime thermocycler 94C 3min -> 8 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 14 cycles of (94C 30sec -> 72C 1min) -> 72C 3min -> 4C hold