Jie:LabNotes/CpgSeq/2008-8-1

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Insertion of spacer in cpg30k_v2.2

PCR amplification of cpg30k_V2.2

reaction system                                                         x48       
 H2O                                                80.7ul           3873.6ul  
 10x buffer                                           10ul              480ul
 MgCl2(25mM)                                           6ul              288ul    
 dNTP(10mM each, NEB)                                  2ul               96ul
 Ap1V4IU       (100uM)                                0.2ul             9.6ul      
 Ap2V4         (100uM)                                0.2ul             9.6ul
 SYBR  reen (50x)                                     0.4ul             19.2ul 
 Jumpstart Taq                                        0.6ul             28.8ul
 template(cpg30KV2.1,20uM)                           0.05ul              2.4ul 
 Total                                                100ul             4800ul
 Reaction program: 94c 2min -> 94c 30sec -> 60C 1.5min -> 72C 1min-> Plate read ->
(94c 30sec -> 60C 1.5min -> 72C 1min)x 22ycles -> 72C 5min -> 15C hold. Ethanol precipitation. Add 125ul ddH2O to amplicons from 24 PCR tubes. Add 15ul 10x Lambda Exonucleus buffer and 8ul lambda Exonucleus, 37C 3h and 90C 10mins. Quiquick column purification. Elute in 60ul ddH2O. yield: ssDNA: 157ng/ul x 60ul.

annealing of cirlehelper

The probe:circlehelper is expected to be 1:1. 
Probe concentration is: 157 ng/ul /(100x330) = 454 nM. 6ul of probe is 28545 x 10^-15 mol.
The volume of CircleHelper(100uM) needed is 0.28545ul.
Annealing system is setup is as follows:
template                         6ul        3         3
10x Ampligase Buffer             3ul        3         3
Circlehelper(100uM)            0.3ul      0.15       0.1
dNTP(10mM)                     0.5ul       0.5
Stoffel fragment(10U/ul)       0.5ul       0.5
Ampligase                        1ul         1
ddH2O                         18.7ul      21.85
94C 3min -> 5 cycles X (94C 30sec -> 60C 10mins) -> 4C .
check product on TBU gel.

File:20080801 after circlehelper ligation.jpg

recalculate: Probe concentration is: 157 ng/ul /(150x330) = 317 nM. 3ul of probe is 9515 x 10^-15 mol.
reset the annealing system is setup is as follows:
template                         3ul       
10x Ampligase Buffer             3ul       
Circlehelper(10uM)               1ul      
dNTP(10mM)                     0.5ul      
Stoffel fragment(10U/ul)       0.5ul      
Ampligase                        1ul      
ddH2O                           21ul      
94C 3min -> 5 cycles X (94C 30sec -> 60C 10mins) -> 4C .
Pool two sets and do PAGE purification of digested product.
 
Quantification: 2ng/ul(40nM)

anneal with AluI oligo and digestion with AluI

1. Add 2 uL of 100 uM RE-AluI-V6 guide oligo in 10ul of template from previous steps. Denature at 95C in PCR machine for 10 min. Turn off the machine and wait for 20 min.
2. Wait until th machine cool to 37C then add 1 uL of AluI restriction enzyme to reaction,incubate for about 2 hours at 37C.
3. Purify the reaction with Minelute columns. Elute in 10ul ddH2O. 

PCR amplification of spacer_107bp

 reaction system                                                  x96        
 H2O                                                80.7ul     7747.2ul  
 10x buffer                                           10ul      960ul
 MgCl2(25mM)                                           6ul      576ul    
 dNTP(10mM each, NEB)                                  2ul      192ul
 spacer_100bp_F(100uM)                               0.2ul      19.2ul
 spacer_100bp_R(100uM)                               0.2ul      19.2ul 
 50x SYBG I                                          0.4ul      38.4ul  
 template(E.coli clone)                              0.1ul       9.6ul
 Jumpstart Taq                                       0.6ul      57.6ul
 Total                                               100ul
 94C 3min -> 8 cycles of (94C 45sec -> 60C 45sec -> 72C 45sec) -> 20 cycles of (94C 30sec -> 68C 45sec -> 72C 30sec)-> 72C 3min -> 4C

File:20080801 spacer PCR.jpg

 Ethanol precipitation. Add 125ul ddH2O to amplicons from 24 PCR tubes. 
 For each 24 PCR amplicons, add 15ul 10x Lambda Exonucleus buffer and 8ul lambda Exonucleus, 37C 3h and 90C 10mins. 
 Quiquick column purification. Elute in 30ul ddH2O.