Alice:LabNotes/2008-11-18
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Cosmic Probe Elongation
Step1: PCR amplification
H2O 50ul SYBR green (50x) 0.4ul primer mix (100uM) 0.2ul Taq master mix(2x) 50ul probe template (cosmic) 0.05ul 94c 2min -> 94c 30sec -> 60C 45sec-> 72C 45sec-> Plate read -> 20 cycles -> 72C 5min -> 15C hold
Step 2: Probe purification with ethanol precipitation
1. pool the 24 reactions into one 16 mL tube, add 240 uL 3M NaOAC, 8 uL GlycoBlue, 6 mL 100% Ethanol. 2. Store at -70C for more than 20 min. 3. Spin at 4000rpm for 15 min at 4C. 4. Remove all liqid, you should see a blue pallette at the bottom of the tube. 5. Add 750 uL cold 75% Ethanol, piette well and transfer to a 1.6 mL tube. 6. Spin at 10000 rpm for 5 min at 4C. 7. Remove all liquid, let the pallette dry for 5 min, resuspend the DNA with 250 uL dH2O.
Step 3: Exonuclease treatment
1. Add 15 uL 10X Exo buffer and 8 uL Lambdo Exonuclease (5K/ml) for total volume of 150uL(each tube). 2. Incubate at 37C for 2 hours followed by 90C for 10 min, and leave it at 4C forever.
Step 4: Purify the reaction with Qiaquick columns
1. for each 150ul tube, add 750 uL of PBI buffer, mix well, load 450 ul each time to column (twice with total of 900 uL) 2. Spin at top speed (14000 rpm) for 1 min 3. Empty the collection tube, add 750 uL wash buffer (PE buffer) to the column 4. Spin at top speed for 1 min 5. Empty the collection tube, spin at top speed for 1 min 6. Carefully transfer the columns to clean 1.6 mL tubes 7. Place the column/tube assemblies to 37C incubator, incubate for 5 min 8. Add 45 uL ddH2O to each column, wait for 1 min, spin at top speed for 1 min. 9. Pool DNAs from the two tubes into one, measure the concentration with Nanodrop.
Step 5: Circularization
Calculate the concentration of template and put 1:2 ratio of template:circlehelper For example: template (956nM) 0.6ul 10x Ampligase Buffer 3ul CircleHelper V_short(1uM) 1.2ul Ampligase(5U/ul) 1ul ddH2O 27ul -------------------------------------------------------- total 32.8ul 94C 2 min -> 55C 20 min Add 2ul of Exo I&III to the two products -> 37C 2hr -> 95C 10min
Step 6: PCR after circlehelper insertion
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 -------------------------------------------------------------------------------- total 100ul
94 2min -> (94 30sec -> 55C 1min -> 72 45 sec)* 22 cycles -> 72C 5min -> 15C hold Purify the products using Qiaquick columns,elute DNA in 30ul ddH2O. Digest the product using Lambda Exonuclease and 10X Exo buffer -> 37C 2hrs -> 90C 10 min Purify it using Qiaquick columns again and use Nanodrop to test the concentration.
Step 7: Spacer insertion
Calculate the concentration of spacer and template, then add them according to 1:1 ratio of template:spacer. Equation: final volume x final concentration = initial volume x initial concentration (final concentration is 20nM)
for example: the final concentration of spacer is 20nM, so 20nM x 50ul = 100nM x amount needed(ul) Add 10ul of each primer, denature at 95C for 5min, and then anneal at 65C for 15 min. the final concentration of template is 20nM, so 20nM x 50ul = template(nM) x amount needed(ul) 20nM x 50ul = 680 nM x 1.5ul Add the following reagents denature at 95C for 5 min, then add the spacer mixture and anneal at 65c for 20 min. template (1) 680nM 1.5 ul spacer mix (100nM each) 20 ul 10x Ampligase Buffer 5 ul Ampligase(5U/ul) 1 ul ddH2O 15.6 ul ---------------------------------------------------------- total 50 ul Add 2ul of ExoI&III 37C for 2hrs -> 94C 10min
Step 8: PCR amplification after spacer insertion
H2O 40ul SYBG 0.4ul primer mix (AP1V4IU+AP2V4) 0.2ul Taq master mix 50ul Template (from previous step) 10ul ---------------------------------------------------------------- total 100ul