Alice:LabNotes/2008-6-27: Difference between revisions
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Step 1: Real time PCR | Step 1: Real time PCR | ||
x12 | x12 | ||
H2O 74.9ul 898.8 ul | H2O 74.9ul 898.8 ul | ||
Line 14: | Line 13: | ||
Total 100ul 1200 ul | Total 100ul 1200 ul | ||
PCR program: 94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> go to step 2 x 21 cycles -> 72C 5min -> 15C hold. | PCR program: 94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> go to step 2 x 21 cycles -> 72C 5min -> 15C hold. | ||
* 48 tubes were pepared* | |||
Results:<br> | |||
[[Image:6-28-08 cosmic probe.jpg]]<br> | |||
Except one curve that went up during very early stage and was eliminated later, otherwise the reaction went very well. | |||
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, | 1. pool the 24 reactions into one 16 mL tube, add 240 uL 3M NaOAC, 8 uL GlycoBlue, | ||
Line 31: | Line 34: | ||
1. Add 15 uL 10X Exo buffer and 8 uL Lambdo Exonuclease (5K/ml) for total volume of 150uL(each tube). | 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. 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 | Step 4: Purify the reaction with Qiaquick columns | ||
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9. Pool DNAs from the two tubes into one, measure the concentration with Nanodrop. | 9. Pool DNAs from the two tubes into one, measure the concentration with Nanodrop. | ||
The experiment was stopped half way because the PE buffer used to purify the samples was not diluted. Therefore everything was washed out and only 1 ng/ul was detected using Nanodrop, which means there was almost no product left and only some background stuff. Next time need to be much more careful with the label. | |||
Latest revision as of 20:13, 30 June 2008
Experiment 1: Cosmic probe preparation[edit]
Step 1: Real time PCR x12 H2O 74.9ul 898.8 ul 10x Platium Taq buffer 10ul 120 ul MgCl2(25mM) 6ul 72 ul 50x SYBG I 0.4ul 4.8 ul dNTP(2.5mM) 8ul 96 ul primer mix (100uM) 0.2ul 2.4 ul template(cosmic 3760) 0.1ul 1.2 ul Platium Taq polymerase 0.4ul 4.8 ul Total 100ul 1200 ul PCR program: 94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> go to step 2 x 21 cycles -> 72C 5min -> 15C hold. * 48 tubes were pepared*
Results:
File:6-28-08 cosmic probe.jpg
Except one curve that went up during very early stage and was eliminated later, otherwise the reaction went very well.
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. Transfer the liquid to two 0.2ml tube.
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 80 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.
The experiment was stopped half way because the PE buffer used to purify the samples was not diluted. Therefore everything was washed out and only 1 ng/ul was detected using Nanodrop, which means there was almost no product left and only some background stuff. Next time need to be much more careful with the label.