Alice:LabNotes/2008-11-18: Difference between revisions

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==Cosmic Probe Elongation==
==Cosmic Probe Elongation==


===Step1: PCR amplification===                               
===PCR amplification===                               
*H2O                                          50ul                         
H2O                                          50ul                         
*SYBR  green (50x)                          0.4ul         
SYBR  green (50x)                          0.4ul         
*primer mix (100uM)                          0.2ul         
primer mix (100uM)                          0.2ul         
*Taq master mix(2x)                          50ul           
Taq master mix(2x)                          50ul           
*probe template (cosmic)                    0.05ul           
probe template (cosmic)                    0.05ul           
   
   
*94c 2min -> 94c 30sec -> 60C 45sec-> 72C 45sec-> Plate read -> 20 cycles -> 72C 5min -> 15C hold
94c 2min -> 94c 30sec -> 60C 45sec-> 72C 45sec-> Plate read -> 20 cycles -> 72C 5min -> 15C hold


===Step 2: Probe purification with ethanol precipitation===
===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.
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.
2. Store at -70C for more than 20 min.
*3. Spin at 4000rpm for 15 min at 4C.
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.
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.
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.
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.
7. Remove all liquid, let the pallette dry for 5 min, resuspend the DNA with 250 uL dH2O.


===Step 3: Exonuclease treatment===
===Exonuclease treatment===
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 hours followed by 90C for 10 min, and leave it at 4C forever.
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===
===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)
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
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
3. Empty the collection tube, add 750 uL wash buffer (PE buffer) to the column
4. Spin at top speed for 1 min
4. Spin at top speed for 1 min
5. Empty the collection tube, 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
6. Carefully transfer the columns to clean 1.6 mL tubes
7. Place the column/tube assemblies to 37C incubator, incubate for 5 min
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.
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.
9. Pool DNAs from the two tubes into one, measure the concentration with Nanodrop.


===Step 5: Circularization===
===Circularization===
Calculate the concentration of template and put 1:2 ratio of template:circlehelper
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


For example:
===PCR after circlehelper insertion===
template (956nM)                          0.6ul
10x Ampligase Buffer                        3ul
H2O                                                40ul       
CircleHelper V_short(1uM)                1.2ul
SYBR  green (50x)                                  0.4ul           
Ampligase(5U/ul)                            1ul
Forward primer AmpRV6.2_2(100uM)                  0.2ul           
ddH2O                                      27ul
Reverse primer AmpFV6.2_2(100uM)                  0.2ul
--------------------------------------------------------
Taq master mix(2x)                                50ul         
total                                    32.8ul
probe template (product after Exo I&III)          10ul  
 
--------------------------------------------------------------------------------
94C 2 min -> 55C 20 min
total                                              100ul
 
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.  
94 2min -> (94 30sec -> 55C 1min -> 72 45 sec)* 22 cycles -> 72C 5min -> 15C hold
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.
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===
===Spacer insertion (11/18/08)===
Calculate the concentration of spacer and template, then add them according to 1:1 ratio of template:spacer.
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
Equation: final volume x final concentration = initial volume x initial concentration
         (final concentration is 20nM)
         (final concentration is 20nM)


for example:
for example:
the final concentration of spacer is 20nM, so 20nM x 50ul =  100nM x amount needed(ul)           
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.
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 = 860 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) 860nM                      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


the final concentration of template is 20nM, so 20nM x 50ul = template(nM) x amount needed(ul)
===PCR amplification after spacer insertion===
                                                20nM x 50ul = 680 nM x 1.5ul
H2O                                                  40ul
 
SYBG                                                0.4ul
Add the following reagents denature at 95C for 5 min, then add the spacer mixture and anneal at 65c for 20 min.
primer mix (AP1V4IU+AP2V4)                          0.2ul
 
Taq master mix                                        50ul
template (1) 680nM                      1.5 ul
Template (from previous step)                        10ul
spacer mix (100nM each)                  20 ul
----------------------------------------------------------------
10x Ampligase Buffer                      5 ul
total                                                100ul
Ampligase(5U/ul)                          1 ul
ddH2O                                  15.6 ul
94C 2min -> 94C 30sec -> 60C 1:30min -> 72C 1min -> plate read -> x22 cycles -> 72C 5 min -> 15C
----------------------------------------------------------
total                                    50 ul
TBE gel result:
 
[[Image:ZhangLab_2 2008-11-20 11hr 39min-.jpg]]
Add 2ul of ExoI&III 37C for 2hrs -> 94C 10min
lane 1: product of spacer insertion step from 11-4-08 & PCR reaction system above
 
lane 2: product of PCR reaction from 11-4-08
===Step 8: PCR amplification after spacer insertion===
lane 3: product of spacer insertion & PCR reaction systems above
H2O                                                  40ul
SYBG                                                0.4ul
primer mix (AP1V4IU+AP2V4)                          0.2ul
Taq master mix                                        50ul
Template (from previous step)                        10ul
----------------------------------------------------------------
total                                                100ul

Latest revision as of 02:07, 25 November 2008

Cosmic Probe Elongation[edit]

PCR amplification[edit]

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

Probe purification with ethanol precipitation[edit]

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.

Exonuclease treatment[edit]

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.

Purify the reaction with Qiaquick columns[edit]

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.

Circularization[edit]

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

PCR after circlehelper insertion[edit]

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.

Spacer insertion (11/18/08)[edit]

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 = 860 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) 860nM                       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

PCR amplification after spacer insertion[edit]

H2O                                                   40ul
SYBG                                                 0.4ul
primer mix (AP1V4IU+AP2V4)                           0.2ul
Taq master mix                                        50ul
Template (from previous step)                         10ul
----------------------------------------------------------------
total                                                100ul

94C 2min -> 94C 30sec -> 60C 1:30min -> 72C 1min -> plate read -> x22 cycles -> 72C 5 min -> 15C

TBE gel result:
File:ZhangLab 2 2008-11-20 11hr 39min-.jpg
lane 1: product of spacer insertion step from 11-4-08 & PCR reaction system above
lane 2: product of PCR reaction from 11-4-08
lane 3: product of spacer insertion & PCR reaction systems above