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

Test Taq@1 digestion[edit]

Test barcode insertion and re-circularization[edit]