Jie:LabNotes/CpgSeq/2008-8-1
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.
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
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 3 Circlehelper(100uM) 0.3ul 0.3 dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase 1ul tube1:94C 3min -> 5 cycles X (94C 30sec -> 60C 10mins) -> 4C . tube2:93C 30min -> 60C 10mins -> add 1ul SLN mix(dNTP 0.8nM) -> 5 cycles X (94C 30sec -> 60C 10mins) check product on TBU gel.