Alice:LabNotes/2008-7-28
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Experiment: Elongate Cosmic probe length[edit]
Circularization procedures of the cosmic 3760 probe can be found on previous Labnotes: [1]
Annealing of Alu-1 oligo[edit]
What is the ratio of guide oligo versus template? --Kun 1. Add 10 uL of 100 uM RE-AluI-V6 guide oligo in 50ul 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 2 uL of AluI restriction enzyme to reaction,incubate for about 4 hours at 37C. 3. Purify the reaction with Minelute columns. Elute in 20ul ddH2O. Nanodrop result: 23.7 ng/ul * 20 ul = 474 ng
Prepare Spacer[edit]
we have two spacer that are waiting to be tested, one is 100 bp FR and other one is 300 bp FR. PCR reaction system setup: x12 H2O 80.1ul 961.2 ul 10x Stoffel buffer 10ul 120 ul MgCl2(25mM) 6ul 72 ul dNTP(10mM each, NEB) 2ul 24 ul primer (Forward) 0.4ul 4.8 ul primer (Reverse) 0.4ul 4.8 ul 50x SYBG I 0.4ul 4.8ul template ( few colonies picked from E coli plate ) Jumpstart Taq 0.6ul 7.2 ul
94c 2min -> 10 cycles (94c 45sec -> 60C 45sec -> 72C 45sec-> Plate read) -> 10 cycles ( 94C 45 sec -> 68C 45 sec -> 72C 45 sec -> plate read) -> 72C 5min -> 15C ethanol precipitation and dilute in 125ul ddH2O. Add 15ul 10x Lambda Exonuclease buffer and 8ul Lambda nuclease(5K/ml). 37C 2h. Quaquick column purification. Elute in 30ul ddH2O. Nanodrop result: 100bp spacer: 155.5ng/ul*30ul= 4665 ng tube 2: 163.3ng/ul*30ul= 4899 ng 300bp spacer: 237.8ng/ul*30ul= 7134 ng tube 2: 240.6ng/ul*30ul= 7218 ng Result: TBE gel of the spacer after PCR: TBU gel of the products (8-5-08): File:ZhangLab 2 2008-07-30 09hr 36min.jpg File:ZhangLab 2 2008-08-05 18hr 22min-1.jpeg
Insert Spacer into Circularized DNA (8/17/08):[edit]
The concentration of cosmic probe after Alu-1 digestion is 23.7 ng/ul, which is 897 nM. The volume will be added into the reaction will be 5ul, which makes it 4.485 umol. 107bp spacer concentration is 155.5 ng/ul, which is 3.001 uM. We need to achieve 1:1 probe/spacer ratio, so the volume of spacer needed is 1.495 ul. 299bp spacer concentration is 237.8 ng/ul, which is 2.019 uM. The volume of spacer needed is 2.221 ul. template 5ul template 5ul spacer(100bp) 1.5ul spacer(300bp) 2.2ul 10x Ampligase Buffer 2.5ul 10x Ampligase Buffer 2.8ul
94C 3min -> 60C 20mins -> add 1ul SLN mix -> 60C 40mins -> add 2ul Exo I/III-> 37C 2h -> 94C 5mins >4C ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP: 0.52ul 0.1ul 0.1ul 0.2ul (2.5mM)0.08ul x20 10.4ul 2ul 2ul 4ul 1.6ul
PCR amplification after insertion of the spacer (8/19/08)[edit]
reaction system x2 H2O 22.4ul 44.8 master mix 25ul 50 primer mix (100uM) 0.2ul 0.4 50x SYBG I 0.4ul 0.4 template 2ul Total 50ul 100 94c 2min -> 94c 30sec -> 60C 1.5min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 1.5min -> 72C 1min)x 35 cycles -> 72C 5min -> 15C hold PCR curve: TBU Gel Result: File:8-19-08.jpg File:8-19-08 PCR after spacer insertion.jpg
Result: The bands of the gel are both at 160bp position, which means the spacer was not successfully inserted into the probe.
Treat circlehelper with Antarctic phosphatase (8/21/08)[edit]
System #1: System #2: circlehelper_V4.2_2_Ida (10uM) 10ul circlehelper_V4.2(10uM) 10ul 10x Antarctic phosphatase buffer 1ul 10x Antarctic phosphatase buffer 1ul Antarctic phosphatase (5U/ul) 1ul Antarctic phosphatase (5U/ul) 1ul 37C 15mins -> 65C 5mins. Annealing system is setup is as follows: template 30ul 10x Ampligase Buffer 5ul Treated Circlehelper(10uM) 1.5ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase 1ul ddH2O 11.5ul 94C 3min -> 5 cycles X (94C 1 min -> 60C 10mins) -> 4C
- Another set of circularized probe is prepared since the previous products are used up.*
circularization of probe using CircleHelper[edit]
reaction system X48 H2O 50ul 2400ul SYBR green (50x) 0.4ul 19.2ul primer mix (100uM) 0.2ul 9.6ul Taq master mix(2x) 50ul 2400ul probe template (cpg30K V2.3) 0.05ul 2.4ul 94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 20 cycles -> 72C 5min -> 15C hold cpg template is used instead since cosmic template is used up. and it is used only to check the accuracy of the protocol. File:8-21-08.jpg
T7 exonuclease digestion[edit]
Purify first 8 tube of product with Qiaquick column, elute in 15ul of ddH2O. Add 7ul T7 exonuclease (10u/ul) and 2 ul of NEBuffer #4. 25C 1h. Purify with Qiaquick column. Elute in 30ul ddH2O. Measure with nanodrop. Result: 28.9ng/ul*15ul
circularization reaction[edit]
template 7ul 10x Ampligase Buffer 2ul CircleHelper V4.2(10uM) 0.6ul reactio system 2 uses CircleHelper V4.2.2 0.6ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase 1ul ddH2O 8.4ul 94C 3min -> 5 cycles X (94C 30sec -> 60C 10mins) -> 4C . check product on TBU gel and column purification of digested product. Gel image after circlehelper insertion: File:2008-08-23 after circlehelper insertion.jpg
annealing with AluI oligo and digestion with ALuI(8/25/08)[edit]
1. Add 1ul NEB buffer2, 1 uL of 100 uM RE-AluI-V6_2_Ida guide oligo in 5ul circularized probe from previous steps. 2. Denature at 95C in PCR machine for 10 mins. Turn off the machine and wait for 20 min. 3. Add 2uL of AluI restriction enzyme to reaction, incubate at 37C for about 2 hours. File:8-25-08 after alu-1 digestion.jpg Note: the left most lane had some urea in the well while loading the gel, so not all the sample traveled down to the bottom of the well when starting the gel, thus might have affected the result of the gel. Theoretically, two lanes should look quite similar based on previous results.