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

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===Step 3: Exonuclease treatment===
===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).
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===
===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)
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===
===Step 5: 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:
For example:
template (956nM)                          0.6ul  
template (956nM)                          0.6ul  
10x Ampligase Buffer                        3ul
10x Ampligase Buffer                        3ul
CircleHelper V_short(1uM)                1.2ul  
CircleHelper V_short(1uM)                1.2ul  
Ampligase(5U/ul)                            1ul
Ampligase(5U/ul)                            1ul
ddH2O                                      27ul
ddH2O                                      27ul
--------------------------------------------------------
--------------------------------------------------------
total                                    32.8ul
total                                    32.8ul


94C 2 min -> 55C 20 min
94C 2 min -> 55C 20 min


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


===Step 6: PCR after circlehelper insertion===
===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


H2O                                                40ul     
  94 2min -> (94 30sec -> 55C 1min -> 72 45 sec)* 22 cycles -> 72C 5min -> 15C hold
SYBR green (50x)                                  0.4ul         
 
Forward primer AmpRV6.2_2(100uM)                  0.2ul         
  Purify the products using Qiaquick columns,elute DNA in 30ul ddH2O.  
Reverse primer AmpFV6.2_2(100uM)                  0.2ul
Digest the product using Lambda Exonuclease and 10X Exo buffer -> 37C 2hrs -> 90C 10 min
Taq master mix(2x)                                50ul       
Purify it using Qiaquick columns again and use Nanodrop to test the concentration.
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.


===Step 7: Spacer insertion===
===Step 7: Spacer insertion===
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)
the final concentration of template is 20nM, so 20nM x 50ul = template(nM) x amount needed(ul)
                                                 20nM x 50ul = 680 nM x 1.5ul
                                                 20nM x 50ul = 680 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.  
Add the following reagents denature at 95C for 5 min, then add the spacer mixture and anneal at 65c for 20 min.
 
template (1) 680nM                      1.5 ul  
template (1) 680nM                      1.5 ul  
spacer mix (100nM each)                  20 ul
spacer mix (100nM each)                  20 ul
10x Ampligase Buffer                      5 ul
10x Ampligase Buffer                      5 ul
Ampligase(5U/ul)                          1 ul
Ampligase(5U/ul)                          1 ul
ddH2O                                  15.6 ul
ddH2O                                  15.6 ul
----------------------------------------------------------
----------------------------------------------------------
total                                    50 ul
total                                    50 ul
 
Add 2ul of ExoI&III 37C for 2hrs -> 94C 10min
Add 2ul of ExoI&III 37C for 2hrs -> 94C 10min


===Step 8: PCR amplification after spacer insertion===
===Step 8: PCR amplification after spacer insertion===
H2O                                                  40ul
H2O                                                  40ul
SYBG                                                0.4ul
SYBG                                                0.4ul
primer mix (AP1V4IU+AP2V4)                          0.2ul
primer mix (AP1V4IU+AP2V4)                          0.2ul
Taq master mix                                        50ul
Taq master mix                                        50ul
Template (from previous step)                        10ul
Template (from previous step)                        10ul
----------------------------------------------------------------
----------------------------------------------------------------
total                                                100ul
total                                                100ul

Revision as of 00:14, 19 November 2008

Cosmic Probe Elongation

Step1: PCR amplification

  • 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

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.

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 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.

Step 5: Circularization

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

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. 
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

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 = 680 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) 680nM                       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

Step 8: PCR amplification after spacer insertion

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