Alice:LabNotes/2008-10-14

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Circularization[edit]

Equation: final volume x final concentration = initial volume x initial concentration

the final concentration of template is 20nM, so 20nM x 30ul = 956nM x amount we need, which is 0.623 ul.

CircleHelper V_short is 100uM, so we take 1 ul of it and add 99ul of H2O to dilute it 100 times to 1uM. The final
concentration of circlehelper is 60nM, so we need 1.8ul.

Add the ligase at 0.02 units/ul ratio. since the total volume is 30ul, we will need to add 0.12 ul of ampligase. 
T4 ligase is 2000U/ul. so we will dilute it to 5U/ul, which is to add 0.5ul of ligase to 199.5ul of H2O.
reaction system 1:
template (956nM)                    0.6ul 
10x Ampligase Buffer                  3ul
CircleHelper V_short(1uM)           1.8ul 
Ampligase(5U/ul)                   0.12ul
ddH2O                                27ul
-----------------------------------------
total                                30ul

94C 2 min -> 55C 20 min
reaction system 2:
template (956nM)                    0.6ul 
10x T4 ligase Buffer                  3ul
CircleHelper V_short(1uM)           1.8ul 
T4 ligase(5U/ul)                   0.12ul
ddH2O                                27ul
-----------------------------------------
total                                30ul

94C 2 min -> 25C 20 min
add 2ul Exo I &III
37C 2hr -> 95 10min -> 4C
Purify using Minelute columns
TBU gel result:
File:ZhangLab 2 2008-10-15 15hr 32min.jpg

PCR after circlehelper insertion(10/15/08)[edit]

reaction systems:
1: circularization of system 1 product from above (before exo I&III digestion)
2: circularization of system 2 product from above (before exo I&III digestion)
3: circularization of system 1 product from above (after exo I&III digestion)
4: circularization of system 1 product from above (after exo I&III digestion)

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
94 2min -> (94 30sec -> 55C 1min -> 72 45 sec)* 15 cycles -> 72C 5min -> 15C hold
TBE gel result:
File:ZhangLab 2 2008-10-16 13hr 28min.jpg
lane 1-4 are the product from reaction systems above, lane 5 is the 10bp ladder

repeat circularization(10/16/08)[edit]

reaction system 1:
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
reaction system 2:
template (956nM)                    0.6ul 
10x T4 ligase Buffer                  3ul
CircleHelper V_short(1uM)           1.2ul 
T4 ligase(5U/ul)                      1ul
ddH2O                                27ul
-----------------------------------------
total                              32.8ul

94C 2 min -> 25C 20 min
Purify the products using qiaquick columns.

Repeat 2: Extending cosmic probe (10/16/08)[edit]

PCR amplification (8 tubes total)                              
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

Purify two tubes of the product using Qiaquick column, and elute the DNA in 30ul ddH2O.

Digest the product using Lambda Exonuclease by adding 3ul of 10x exonuclease buffer, and 2ul of Lambda Exo.
Purify it using Qiaquick columns again.

Nanodrop result: 52.2ng/ul, which is 1.1uM.

Repeat 2: circularization (10/17/08)[edit]

reaction system 1:
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

reaction system 2:
template (956nM)                    0.6ul 
10x T4 ligase Buffer                  3ul
CircleHelper V_short(1uM)           1.2ul 
T4 ligase(5U/ul)                      1ul
ddH2O                                27ul
-----------------------------------------
total                              32.8ul

94C 2 min -> 25C 20 min

Add 2ul of Exo I&III to the two products -> 37C 2hr -> 95C 10min

Repeat 2: PCR after circlehelper insertion(10/21/08)[edit]

systems setup:
1: product of repeat circularization(10/16/08)-reaction system 1 after treated with Exo I&III
2: product of repeat circularization(10/16/08)-reaction system 2 after treated with Exo I&III
3: product of Repeat 2: circularization (10/17/08)-reaction system 1 after treated with Exo I&III
4: product of Repeat 2: circularization (10/17/08)-reaction system 2 after treated with Exo I&III

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 

94 2min -> (94 30sec -> 55C 1min -> 72 45 sec)* 22 cycles -> 72C 5min -> 15C hold

Gel results of the experiments under different conditions (10/20-21):[edit]

TBU gel result of the circularization products (see above for protocol used): 
File:ZhangLab 2 2008-10-20 11hr 04min.jpg
1: repeat 2-PCR product of cosmic probe 
2: repeat 2-digested PCR product using lambda Exo
3: repeat circularization(10/16/08)-reaction system 1
4: repeat circularization(10/16/08)-reaction system 2
5: repeat circularization(10/16/08)-reaction system 1 after treated with Exo I&III
6: repeat circularization(10/16/08)-reaction system 2 after treated with Exo I&III
7: Repeat 2: circularization (10/17/08)-reaction system 1
8: Repeat 2: circularization (10/17/08)-reaction system 2
9: Repeat 2: circularization (10/17/08)-reaction system 1 after treated with Exo I&III
10: Repeat 2: circularization (10/17/08)-reaction system 2 after treated with Exo I&III
TBE gel of the PCR results:
File:ZhangLab 2 2008-10-21 15hr 37min.jpg
(see sections  Repeat 2: Extending cosmic probe (10/16/08) and  
Repeat 2: PCR after circlehelper insertion(10/21/08) above for detailed protocol)

1: PCR product of repeat circularization(10/16/08)-reaction system 1 after treated with Exo I&III
2: PCR product of repeat circularization(10/16/08)-reaction system 2 after treated with Exo I&III
3: PCR product of Repeat 2: circularization (10/17/08)-reaction system 1 after treated with Exo I&III
4: PCR product of Repeat 2: circularization (10/17/08)-reaction system 2 after treated with Exo I&III
5: PCr product of Repeat 2: Extending cosmic probe (10/16/08)

Note: the PCR curve never shoot up for lane 2 and 4. Lane 3 was digested with lambda Exonuclease and shoot up at 10th cycle, 
while lane 1 was treated with T7 exonuclease and shoot up around 15th cycle.