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 1.5ul NEB buffer 1, 2 uL of 100 uM RE-AluI-V6 guide oligo in 10ul of template from previous steps(ratio is 500:1). Denature at 95C in PCR machine for 10 min. Turn off the machine and wait for 20 min.
2. Add 2.5uL of AluI restriction enzyme to reaction, incubate at 37C for about 2 hours.
3. Purify the reaction with Minelute columns. Elute in 10ul ddH2O. 

File:20080805 after AluI digestion.jpg

Minelute column purification. Elute in 10ul ddH2O. Quantification: 6.5ng/ul x 10ul

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.jpg20080801_107bp_spacer_PCR(The PCR fragment is 157bp. The spacer length is 107bp)

 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.
 yield: 156.7ng/ul x 30ul.

set up spacer insertion system

template(5.6ng/ul)               5ul       
10x Ampligase Buffer             1ul       
spacer(156ng/ul)                 5ul      
94C 3min -> 65C 20mins -> add 1ul SLN mix -> 65C 20mins -> add 2ul Exo I/III-> 37C 2h -> 94C 5mins -> 4C .

File:20080805 after spacer insertion.jpg20080804_after spacer insertion

PCR amplification after insertion of spacer_107bp

 reaction system                                                     
 H2O                                                18.4ul     
 iProof 2x mix                                        25ul      
 spacer_100bp_F(100uM)                               0.2ul      
 spacer_100bp_R(100uM)                               0.2ul      
 50x SYBG I                                          0.2ul      
 template                                              6ul      
 Total                                                50ul
 98C 2min -> 22 cycles of (98C 10sec -> 63C 20sec -> 72C 20sec) -> 72C 3min -> 4C

File:20080804 PCR after spacer insertion.jpg20080804_PCR after spacer insertion

 I did not get expected size. I think it is because of high spacer concentration in the spacer insertion system.
 I ordered a new AluI oligo on 08/11/08. 
 AluI_oligo_2_Ida: cctcgataacatctgcagctgaataggaacgatgag

redo the anneal with AluI oligo and digestion with AluI 2008-08-12

1. Add 3ul NEB buffer 2, 2 uL of 100 uM RE-AluI-V6_2_Ida guide oligo in 20ul circularized probe (2ng/ul) from previous steps(ratio is 250:1). 
2. Denature at 95C in PCR machine for 10 min. Turn off the machine and wait for 20 min.
2. Add 5uL of AluI restriction enzyme to reaction, incubate at 37C for about 2 hours.

File:20080813 Gel cut after AluI digestion.jpg20080813_Gel cut after AluI digestion

File:20080813 quantification of the product after AluI digestion.jpg20080813_quantification of the product after AluI digestion

Gel purification of the 160bp size. yield: 1ng/ul (19nM)x 15ul

File:20080814 quantification of probe and spacer.jpg20080814_quantification of probe and spacer

set up spacer insertion system

 Dilute the 156ng/ul(3uM) spacer to 1ng/ul
 template(1ng/ul;19nM)            6.5ul       
 10x Ampligase Buffer             1.5ul       
 spacer(1ng/ul;19nM)              6.5ul      
94C 3min -> 60C 20mins -> add 1ul SLN mix (dNTP 0.2mM) -> 65C 20mins -> add 2ul Exo I/III-> 37C 2h -> 94C 5mins -> 4C .

PCR amplification after insertion of spacer_107bp 2008-08-14

 reaction system                                               x2        
 H2O                                                22.4ul     44.8  
 iProof 2x mix                                        25ul       50
 spacer_100bp_F(100uM)                               0.2ul      0.4
 spacer_100bp_R(100uM)                               0.2ul      0.4
 50x SYBG I                                          0.2ul      0.4
 template                                              2ul      
 Total                                                50ul      100
 98C 2min -> 22 cycles of (98C 10sec -> 63C 20sec -> 72C 20sec) -> 72C 3min -> 4C

File:20080814 PCR after insertion of spacer.jpg20080814_PCR after insertion of spacer

 I realized that the probe, after the Stoffel fragment amplification and ligation of cirlce helper, will be converted into a 
double-strand cirlce that has two ALuI restriction site(one is on linker and the other is on cirlchelper). The following AluI digestion will cut
the probe at two sites and produce a 90-mer and a 70-mer pieces. The 160-mer on the gel is the
reverse strand which was cut by AluI on the circlhelper site.
 To confirm the hypothesis, I digested the circularized cpg30k_V2.2 (with circlehelper) by AluI without AluI-oligo.
 File:20080817-digestion without AluI oligo.jpg
 The image showed that the AluI digested the circle without AluI-oligo, which means that the circularization with circlehelper may form a double-stranded circular DNA (180-mer cutted from TBU gel). 
 I ordered a new circlehelper_V4.2_2_Ida, which replaced the AluI site with 'ATCA':
 5'- AAC AGT GCT CTT CCA GTC TAC ATC AGA ATT CTA GCC TCA TGC GTA TCC GAT C -3'

annealing of cirlehelperV4.2_2_Ida (2008-08-18)

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:
                                tube1     tube2
template                         6ul        6        
10x Ampligase Buffer             1ul        1        
Circlehelper(100uM)            0.3ul      0.3       
dNTP(10mM)                     0.5ul      
Stoffel fragment(10U/ul)       0.5ul      
Ampligase                        1ul       H2O 2ul
                                         
tube1:94C 3min -> 5 cycles X (94C 1 min -> 60C 10mins) -> 4C .
tube2:94C 3min -> 60C 5mins -> add 1ul SLN mix(dNTP 0.8nM) -> 60C 5min ->4 cycles X (94C 1 min -> 60C 10mins)
check product on TBU gel.

File:20080819 annealing of new cirlcehelp.jpg

do PAGE purification of digested product.
  
Quantification: ng/ul(40nM)

anneal with AluI oligo and digestion with AluI 2008-08-19

1. Add 3ul NEB buffer 2, 2 uL of 100 uM RE-AluI-V6_2_Ida guide oligo in 20ul circularized probe (2ng/ul) from previous steps(ratio is 250:1). 
2. Denature at 95C in PCR machine for 10 min. Turn off the machine and wait for 20 min.
2. Add 5uL of AluI restriction enzyme to reaction, incubate at 37C for about 2 hours.