Dinh 2011/NOTES/2011-9-1
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Test circularization #3[edit]
Dr. Zhang's notes:
Reaction: Linear template 30 ul Ampligase buffer (10x) 5 ul Circle helper (10uM) 0.25 ul dNTP (10mM) 0.5 ul Stoffel (10U/ul) 0.5 ul Ampligase (5U/ul) 1 ul H2O 12.5 ul =================================== 50 ul Program: 94 C 3min -> (96C 30s -> 60C 10 min)x5
Suggested running gel before adding Exo I and III. Suggested adding dNTPs and Stoffel to fill in possible oligo gaps Amount of dNTP was 5x10^-9 moles
Splint to probes ratio
Amount of circle helper was 2.5x10^-12 moles Amount of template: (splint to template) # moles Concentration 1:1 2.50x10^-12 0.083 uM (3.54 ng/ul) 2:1 1.25x10^-12 0.041 uM (1.75 ng/ul) 1:2 5.00x10^-12 0.167 uM (7.08 ng/ul)
Convert probes X uM to ng/ul: X * (10^-6 moles/L) * (MW kg/moles) * (10^-6 L/ul) * (10^12 ng/kg)
Dr. Zhang's probes: ~ MW Template ~ 140 nt (single stranded) = 42.6 kDa (kg/moles) DMR330K probes: ~ MW Template ~ 150 nt (single stranded) = 45.6 kDa (kg/moles)
Set up for DMR330K probes:
Reaction: Linear template 30 ul Ampligase buffer (10x) 5 ul Circle helper (10uM) 0.25 ul dNTP *(1mM) 0.5 ul Stoffel (10U/ul) 0.5 ul Ampligase (5U/ul) 1 ul H2O 12.5 ul =================================== 50 ul
*Reduce the amount of dNTP to add: Use 100 moles of dNTP per 1 moles of template - 5x10^-10 moles of dNTP Set up 3 different concentrations for template: Amount of template: (splint to template) # moles Concentration 1:2 5.00x10^-12 0.167 uM (7.57 ng/ul) 1:1 2.50x10^-12 0.083 uM (3.78 ng/ul) 2:1 1.25x10^-12 0.041 uM (1.89 ng/ul)
Template dilution: Lambda exo digested probes = 98ng/ul 98 ng/ul * X ul = 7.57 * 70 ul 1) Add 5.41 ul probes (98ng/ul) + 64.59 ul H2O -> 7.57 * 70 ul 2) Take 30 ul probes (7.57 ng/ul) + 30 ul H2O -> 3.78ng/ul * 60 ul (1 with enzyme and 1 with no enzyme as control) 3) Take 7.5 ul probes (7.57 ng/ul) + 22.5 ul H2O -> 1.89ng/ul * 30 ul For running at 3 different temperatures: 1) Add 16.2 ul of 98ng/ul probes + 193.8 ul H2O (#1) 2) Add 60ul of #1 + 60 ul of H2O (30+30+30+30) 3) Add 22.5 ul of #1 + 67.5 ul of H2O
Master mix: 1x 10x H2O 12.5 -> 125 Splint (10uM) 0.25 -> 2.5 dNTP (*0.5mM) 0.50 -> 5.0 Ampligase buffer (10x) 5 -> 50 Stoffel (10U/ul) 0.50 -> 5.0 =======================================> Take out 18.75 ul for no-enzyme control Ampligase (5U/ul) 1.00 -> 9.0 (only 9x now) ------------------------------------------ Add 19.75 ul per reaction *Reduced amount of dNTPs by 1/2.
Program: 94 C 3min -> (94C* 30s -> Gradient (55C-60C) 10 min)x5 Place a set at 55,58,and 60C each * I forgot to change it to 96C.
Pipette 2.5 ul after 3 cycles, 4 cycles, and 5 cycles for running gel.
Run TBU gel. At 4 cycles: File:ZhangLab 2 2011-09-01 Circularization -3 - 4cycles.jpg
At 5 cycles: File:ZhangLab 2 2011-09-01 Circularization -3 - 5cycles.jpg
Results Still too high long concatemers to circle product ratio -> especially when the splint to probes ratio is 1:2, reduce the ratio further Why three bands from template? -> need to do a no stoffel control.
Test circularization #4[edit]
Template dilution: Lambda exo digested probes = 98ng/ul 98 ng/ul * X ul = 3.78 * 110 ul 1) Add 4.2 ul probes (98ng/ul) + 105.8 ul H2O -> 3.78 * 110 ul (30 test + 30 no ampligase* + 30 no stoffel, no ampligase) 2) Take 15 ul probes (3.78 ng/ul) + 15 ul H2O -> 1.89 ng/ul * 30 ul (30 test) * Should've been a no stoffel control :(
For running at 3 different temperatures: 1) Add 12.6 ul of 98ng/ul probes + 317.4 ul H2O (#1) 2) Add 45ul of #1 + 45 ul of H2O
Cut 3 strips of 4 tubes.
Mix A : 1x 13x H2O 12.5 -> 162.5 Splint (10uM) 0.25 -> 3.25 dNTP (1mM) 0.50 -> 6.5 Ampligase buffer (10x) 5 -> 65 ======================================== 17.75 each to 12 tubes
First 6 tubes: Enzyme Mix 1 : 1x 7x Ampligase (5U/ul) 1.00 -> 7.0 Stoffel (10U/ul) 0.50 -> 3.5 ======================================== 1.5 each to 6 tubes Next 3 tubes: Enzyme Mix 2 : (Stoffel only) 4x H2O 1.00 -> 4.0 Stoffel (10U/ul) 0.50 -> 2.0 ======================================== 1.5 each to 3 tubes
Next 3 tubes: No Enzyme Mix 3 : H2O 1.50 ======================================== 1.5 each to 3 tubes
Program: 94 C 3min -> (96C 30s -> Gradient (55C-60C) 10 min)x5 Place a set at 55,58,and 60C each
Pipette 2.5 ul after 1, 2, 3, 4, and 5 cycles for running gel.
Cycles 1-4 at 55C File:ZhangLab 2 2011-09-01 Test 4 circularization 55C cycles 1-4 and at cycle 5 no L, no SL.jpg
Cycles 1-4 at 60C File:ZhangLab 2 2011-09-01 Test 4 circularization 60C cycles 1-4 and at cycle 5 no L, no SL.jpg
Compate 55C, 58C, and 60C File:ZhangLab 2 2011-09-01 Compare 55,58,60.jpg