Alice:LabNotes/2008-9-3
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Experiment: Extending the cosmic probe length[edit]
previous steps and the over-view of the experiment can be found through this link [[1]]
T7 exonuclease digestion[edit]
obtained 48 reactions tubes from Ida on cpg30K V2.2 1. The tubes were purified using ethanol purification, and diluted in 100ul H2O for every 24 reactions. 2. Add ratio of 0.5ul/tube amount of T7, which is 12ul of T7 and 11ul of NEBuffer #4 to each tube(24 reactions). 3. 25C for 2 hrs. 4. Qiaquick column purification, elute in 30ul of H2O. 5. Measure with Nanodrop. Nanodrop result: 125.8ng/ul TBE gel of the product after PCR and T7 digestion: File:9-4-08.jpg
circularization reaction[edit]
the concentration of the template from last step is 125.8ng/ul, so it is equivalent to 125.8/144/330=2.64uM
reaction system 1: 1:1 ratio of template:circleHelper reaction system 2: 1:3 ratio of template:circleHelper reaction system 1: template (264nM) 2ul 10x Ampligase Buffer 2ul CircleHelper V4.2(1uM) 0.5ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase(5U/ul) 0.5ul ddH2O 14ul reaction system 2: template (264nM) 2ul 10x Ampligase Buffer 2ul CircleHelper V4.2(1uM) 1.5ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase(5U/ul) 0.5ul ddH2O 13ul 94C 2 min -> 55C 20 min
TBU gel after the circularization reaction: File:9-5-08.jpg add 1.5ul Exo I &III + 10ul of product from reaction system 1 37C 2hr -> 95 10min -> 4C Purify using Minelute columns
9/9/08: add 2.5ul Exo I&III + 20ul of product from reaction system 2 37C 2hr -> 95 10min -> 4C Purify using Minelute columns
PCR (9/9/08)[edit]
H2O 40ul SYBR green (50x) 0.4ul Forward primer AmpRV6.2_2(100uM) 0.2ul Reverse primer AmpFV6.2_2(100uM) 0.2ul Taq master mix(2x) 50ul probe template (product after Exo I&III) 10ul 94 2min -> (94 30sec -> 55C 1min -> 72 45 sec)* 15 cycles -> 72C 5min -> 15C hold File:9-9-08 PCR after Exo I&III.jpg TBU gel of each step:File:9-9-08 after pcr.jpg
T7 exonuclease digestion (9/9/08)[edit]
Add 2ul of T7 exonuclease to the PCR product 25C 2hr -> 4C Purify with Qiaquick columns, and measure the concentration using Nanodrop to determine the amount of spacer needed. Nanodrop result: 27.4ng/ul * 30ul
Insert Spacer into Circularized DNA (9/10/08)[edit]
27.4ng / (160*330) = 510nM, 5ul of template makes it 2.55 umol. 107bp spacer concentration is 156.7 ng/ul, which is 4.4 uM. We need to achieve 1:1 probe/spacer ratio, so the volume of spacer needed is 0.6 ul.
template 5ul 10x Ampligase Buffer 2ul 107bp Spacer 0.6ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase(5U/ul) 0.5ul ddH2O 11ul (94C 2min -> 60C 20min ) x4 cycles add 2ul Exo I/III-> 37C 2h -> 94C 5mins >4C
TBU gel of the results: File:9-11-08 after spacer insertion.jpg
PCR amplification after spacer insertion[edit]
H2O 70.6ul 10x Stoffel buffer 10ul MgCl2(25mM) 6ul dNTP(10mM each, NEB) 2ul Primer mix 100uM (Ap1V4IU +Ap2V4) 0.4ul 50x SYBG I 0.4ul template(from last step) 10ul Jumpstart Taq 0.6ul
94c 2min -> ( 94c 30sec -> 60C 1.5min -> 72C 1min-> Plate read -> ) x35 cycles -> 72C 5min -> 15C hold TBE gel after PCR: File:9-11-08 PCR after spacer insertion.jpg File:ZhangLab 2 2008-09-12 10hr 37min.jpg