Jie:LabNotes/ASE/2008-2-29

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Optimization of the padlock captureing efficiency[edit]

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 strand displacement.
   Tube 2 decrease the dNTP concentration with gradient 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(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     x6     tube 4
 H2O                              5.5ul         5.5ul          5.5ul       5.5ul      5.5ul     5.5ul     33ul     1ul 
 10x Ampligase Buffer               1ul           1ul            1ul         1ul        1ul       1ul      6ul     1ul
 probe (CES22K,9.3ng/ul)          1.5ul         1.5ul          1.5ul       1.5ul      1.5ul     1.5ul      9ul     6ul
 Jurkat Genomic DNA(100ng/ul)       2ul           2ul            2ul         2ul        2ul       2ul     12ul     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 -> add 1ul ddH2O -> 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
 prepare 5x(ddH2O 3ul, Ligase Buffer 1.5ul, Ligase 1.5ul, Amplitaq 3ul, mix and aliquot into 3 tubes,
then add dNTP 2ul with different concentration respectively.

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 100uM  

PCR[edit]

PCR reaction system

 reaction system                                                         x14
 H2O                                                30.6ul            428.4ul                   
 10x JumpStartTaq Buffer                               5ul               70ul                   
 MgCl2(25mM)                                           3ul               42ul 
 dNTP(10mM)                                            2ul               28ul                  
 AmpSolV6F2/R2 mix(10uM)(no tail)                      2ul               28ul                 
 50x SYBG I                                          0.4ul              5.6ul                   
 JumpStartTaq                                          2ul               28ul                   
 Products of Circluarization                           5ul               70ul                   
 Total                                                50ul              700ul

Run the program on Opticon4 realtime thermocycler 94C 3min -> 24 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 72C 3min -> 4C hold If the product will be sequenced, the primers with tail are used and two set of PCR program is used. See Labnote on 21 Feb.

Primer:

 AmpFV6.2: CTGCAGATGTTATCGAGGTC Tm=58
 AmpRv6.2: TGCAGCTGAATAGGAACGAT Tm=60

Result:

 From left to right: purple is T2, dark yellow is T1, light blue is T3, yellow is T4, green is S2, red is S1,dark blue is S3. 
 

File:2008 02 29 capture optimization 1.png Image:2008_02_29_capture_optimization_1

File:2008 02 29 capture optimization 2.png Image:2008_02_29_capture_optimization_2