Alice:LabNotes/2008-7-28

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Experiment: Elongate Cosmic probe length[edit]

Circularization procedures of the cosmic 3760 probe can be found on previous Labnotes: [1]

Annealing of Alu-1 oligo[edit]

What is the ratio of guide oligo versus template? --Kun
1. Add 10 uL of 100 uM RE-AluI-V6 guide oligo in 50ul of template from previous steps. Denature at 95C in PCR machine for 10 min. Turn off the machine and wait for 20 min.
2. Wait until th machine cool to 37C then add 2 uL of AluI restriction enzyme to reaction,incubate for about 4 hours at 37C.
3. Purify the reaction with Minelute columns. Elute in 20ul ddH2O. 

Nanodrop result: 23.7 ng/ul * 20 ul = 474 ng

Prepare Spacer[edit]

we have two spacer that are waiting to be tested, one is 100 bp FR and other one is 300 bp FR.

PCR reaction system setup:                                           x12         
H2O                                                 80.1ul         961.2 ul
10x Stoffel buffer                                    10ul           120 ul
MgCl2(25mM)                                            6ul            72 ul
dNTP(10mM each, NEB)                                   2ul            24 ul
primer (Forward)                                     0.4ul           4.8 ul
primer (Reverse)                                     0.4ul           4.8 ul
50x SYBG I                                           0.4ul            4.8ul
template                                           ( few colonies picked from E coli plate )       
Jumpstart Taq                                        0.6ul           7.2 ul
94c 2min -> 10 cycles (94c 45sec -> 60C 45sec -> 72C 45sec-> Plate read) -> 10 cycles ( 94C 45 sec -> 68C 45 sec -> 72C 45 sec ->
plate read) -> 72C 5min -> 15C

ethanol precipitation and dilute in 125ul ddH2O. Add 15ul 10x Lambda Exonuclease buffer and 8ul Lambda nuclease(5K/ml). 37C 2h.
Quaquick column purification. Elute in 30ul ddH2O. 
Nanodrop result: 
100bp spacer: 155.5ng/ul*30ul=  4665 ng         tube 2: 163.3ng/ul*30ul= 4899 ng
300bp spacer: 237.8ng/ul*30ul=  7134 ng         tube 2: 240.6ng/ul*30ul= 7218 ng

Result:
TBE gel of the spacer after PCR:                            TBU gel of the products (8-5-08):  
File:ZhangLab 2 2008-07-30 09hr 36min.jpg                         File:ZhangLab 2 2008-08-05 18hr 22min-1.jpeg

Insert Spacer into Circularized DNA (8/17/08):[edit]

The concentration of cosmic probe after Alu-1 digestion is 23.7 ng/ul, which is 897 nM. The volume will be added
into the reaction will be 5ul, which makes it 4.485 umol.
107bp spacer concentration is 155.5 ng/ul, which is 3.001 uM. We need to achieve 1:1 probe/spacer ratio, so the volume of
spacer needed is 1.495 ul. 
299bp spacer concentration is 237.8 ng/ul, which is 2.019 uM. The volume of spacer needed is 2.221 ul.

template                          5ul                   template                          5ul
spacer(100bp)                   1.5ul                   spacer(300bp)                   2.2ul        
10x Ampligase Buffer            2.5ul                   10x Ampligase Buffer            2.8ul
94C 3min -> 60C 20mins -> add 1ul SLN mix -> 60C 40mins -> add 2ul Exo I/III-> 37C 2h -> 94C 5mins >4C                        

                                                                      ddH2O     Ligase Buffer   Ligase     Amplitaq    dNTP           
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP:      0.52ul         0.1ul       0.1ul       0.2ul    (2.5mM)0.08ul 
                                                           x20        10.4ul           2ul         2ul         4ul      1.6ul

PCR amplification after insertion of the spacer (8/19/08)[edit]

reaction system                                               x2        
H2O                                                22.4ul     44.8  
master mix                                           25ul       50
primer mix (100uM)                                  0.2ul      0.4                            
50x SYBG I                                          0.4ul      0.4
template                                              2ul      
Total                                                50ul      100

94c 2min -> 94c 30sec -> 60C 1.5min -> 72C 1min-> Plate read -> (94c 30sec -> 60C
1.5min -> 72C 1min)x 35 cycles -> 72C 5min -> 15C hold

PCR curve:                                                  TBU Gel Result: 
File:8-19-08.jpg               File:8-19-08 PCR after spacer insertion.jpg

Result: The bands of the gel are both at 160bp position, which means the spacer was not successfully inserted into the probe.

Treat circlehelper with Antarctic phosphatase (8/21/08)[edit]

 System #1:                                System #2:
 circlehelper_V4.2_2_Ida (10uM)      10ul  circlehelper_V4.2(10uM)         10ul        
 10x Antarctic phosphatase buffer    1ul   10x Antarctic phosphatase buffer    1ul
 Antarctic phosphatase (5U/ul)       1ul   Antarctic phosphatase (5U/ul)       1ul
 37C 15mins -> 65C 5mins.

Annealing system is setup is as follows:
                                   
template                          30ul              
10x Ampligase Buffer               5ul            
Treated Circlehelper(10uM)       1.5ul             
dNTP(10mM)                       0.5ul      
Stoffel fragment(10U/ul)         0.5ul      
Ampligase                          1ul
ddH2O                           11.5ul      
                                         
94C 3min -> 5 cycles X (94C 1 min -> 60C 10mins) -> 4C
  • Another set of circularized probe is prepared since the previous products are used up.*

circularization of probe using CircleHelper[edit]

reaction system                                           X48              
H2O                                       50ul          2400ul              
SYBR  green (50x)                        0.4ul          19.2ul
primer mix (100uM)                       0.2ul          9.6ul                 
Taq master mix(2x)                        50ul          2400ul 
probe template (cpg30K V2.3)             0.05ul         2.4ul

94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 20 cycles -> 72C 5min -> 15C hold

cpg template is used instead since cosmic template is used up. and it is used only to check the accuracy of the protocol.

File:8-21-08.jpg

T7 exonuclease digestion[edit]

Purify first 8 tube of product with Qiaquick column, elute in 15ul of ddH2O. Add 7ul T7 exonuclease (10u/ul) and 2 ul of
NEBuffer #4. 25C 1h. Purify with Qiaquick column. Elute in 30ul ddH2O. Measure with nanodrop. 

Result: 28.9ng/ul*15ul

circularization reaction[edit]

template                          7ul
10x Ampligase Buffer              2ul  
CircleHelper V4.2(10uM)         0.6ul  reactio system 2 uses CircleHelper V4.2.2  0.6ul
dNTP(10mM)                      0.5ul
Stoffel fragment(10U/ul)        0.5ul
Ampligase                         1ul
ddH2O                           8.4ul

94C 3min -> 5 cycles X (94C 30sec -> 60C 10mins) -> 4C .
check product on TBU gel and column purification of digested product.
Gel image after circlehelper insertion:
File:2008-08-23 after circlehelper insertion.jpg

annealing with AluI oligo and digestion with ALuI(8/25/08)[edit]

1. Add 1ul NEB buffer2, 1 uL of 100 uM RE-AluI-V6_2_Ida guide oligo in 5ul circularized probe from previous steps.
2. Denature at 95C in PCR machine for 10 mins. Turn off the machine and wait for 20 min.
3. Add 2uL of AluI restriction enzyme to reaction, incubate at 37C for about 2 hours.
File:8-25-08 after alu-1 digestion.jpg
Note: the left most lane had some urea in the well while loading the gel, so not all the sample traveled down to the bottom
of the well when starting the gel, thus might have affected the result of the gel. Theoretically, two lanes should look quite
similar based on previous results.