Alice:LabNotes/2008-10-28

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spacer insertion[edit]

For the product and procedure from previous steps, please follow this link (10/14/08 LabNote): 
[[1]]
template used:
(1) PCR product of repeat: circularization(10/16/08)-reaction system 1 after treated with Exo I&III
(2) PCR product of Repeat 2: circularization (10/17/08)-reaction system 1 after treated with Exo I&III 
PCR results from lane 1 and 3 (see section Repeat 2: PCR after circlehelper insertion(10/21/08) from link above) was purified
with Qiaquick columns,elute DNA in 30ul ddH2O. 
Digest the product using Lambda Exonuclease by adding 3ul of 10x exonuclease buffer, 2ul of Lambda Exo. 37C 2hrs -> 90C 10 min
Purify it using Qiaquick columns again and use Nanodrop to test the concentration.
Nanodrop result: (1) 5.7 ng/ul = 119nM             (2) 10.1 ng/ul = 680 nM <-this is a calculation error!

Note: this proves that digest the PCR product with exonuclease gives about twice as much %yield as using T7 exonuclease.
Equation: final volume x final concentration = initial volume x initial concentration
*previous experiments show that ligation process works better with low concentration of template and primers*

The amount of insertion spacer oligo ordered was 2nmol, and the reverse arm was 4.8nmol. Diluting them using 200ul and 480ul
of ddH2O respectively yields final concentration of 10uM. Dilute the spacer to 10nM since the PCR product concentration
is quite low (we need 1:1 ratio of spacer:template)
Prepare the spacer: Add 1ul of spacer to 99ul of ddH2O to dilute them to 100nM
                    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 10min.

Prepare the template: the final concentration of template is 20nM, so 20nM x 50ul = template(nM) x amount needed(ul)
                      template (1) needs 20nM x 50ul = 119 nM x 8.4ul
                      template (2) needs 20nM x 50ul = 680 nM x 1.5ul
                      Add 8.4ul of template(1) and 14.1ul of ddH2O;
                      Add 1.5ul of template(2) and 21ul of ddH2O; 
                      denature at 95C for 5min, quickly add the spacer prepared from last step, anneal at 65C for 10 min  
                      Add the remaining reagent in the chart below (color green) and anneal at 60C for 20min.
spacer insertion:  

reaction system 1:
template (1) 119nM                  8.4 ul 
spacer mix (100nM each)              20 ul 
10x Ampligase Buffer                  5 ul
Ampligase(5U/ul)                      1 ul
dNTP(10mM)                            1 ul            
Stoffel fragment(10U/ul)            0.5 ul
ddH2O                              14.1 ul
------------------------------------------
total                                50 ul 

reaction system 2:
template (2) 680nM (211nM actually) 1.5 ul
spacer mix (100nM each)              20 ul
10x Ampligase Buffer                  5 ul
Ampligase(5U/ul)                      1 ul
dNTP(10mM)                            1 ul 
Stoffel fragment(10U/ul)            0.5 ul
ddH2O                                21 ul
------------------------------------------
total                                50 ul

Add 2ul of Exo I&III -> 37C 2hr -> 95C 10min
purify the products with Qiaquick columns
File:ZhangLab 2 2008-10-31 13hr 37min.jpg
1: template (1) before spacer insertion
2: template (2) before spacer insertion
3: reaction system 1 after spacer insertion
4: reaction system 2 after spacer insertion
5: reaction system 1 after spacer insertion and ExoI&III
6: reaction system 2 after spacer insertion and ExoI&III
PCR amplification after spacer insertion (10/30/08):
H2O                                                   40ul              
Primer mix 100uM (Ap1V4IU +Ap2V4)                    0.2ul             
50x SYBG I                                           0.4ul         
template(from last step)                              10ul         
Taq 2x master mix                                     50ul
94c 2min -> ( 94c 30sec -> 60C 1.5min -> 72C 1min-> Plate read -> ) x25 cycles -> 72C 5min -> 15C hold
File:ZhangLab 2 2008-10-31 14hr 03min.jpg
1: PCR product of cosmic probe 
2: reaction system (1) after PCR
3: reaction system (2) after PCR

Diagnosing spacer insertion efficiency (10/31/08)[edit]

The above gel images seems somehow problematic before the cosmic probe product looks weird, it should have bands around 145bp
region. Therefore I am going to repeat and procedures and test if the spacer insertions are able to anneal with each other. 
Then put them into the template using the same protocol as Circlehelper insertion.
 
Add 1ul of spacer to 99ul of ddH2O to dilute them to 100nM
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 20min.

several results of the TBE gel from above system had problems such as bad gel or not loading enough samples, therefore they 
are not posted.