Library/NOTES/2010-10-22: Difference between revisions

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  -From DNA conc. by PAGE quantification, DNA samples were divided in two subgroups  
  -From DNA conc. by PAGE quantification, DNA samples were divided in two subgroups  
  - '''Gr.1 (*) Conc.>1.0ng/ul:''' MG1, MG2, MG4, MG5, MG9, MG15 & MG32
  - '''Gr.1 (*) Conc.>1.0ng/ul:''' MG1, MG2, MG4, MG5, MG9, MG15 & MG32
  - '''Gr.2 (**) Conc.<1.0ng/ul:///''' MG8, MG13, MG14 & MG16
  - '''Gr.2 (**) Conc.<1.0ng/ul:''' MG8, MG13, MG14 & MG16
  - Sample MG12 didn't show the product in the gel then it was repeated again with larger amount of sheared DNA, 65ul in end repaired step.
  - Sample MG12 didn't show the product in the gel then it was repeated again with larger amount of sheared DNA, 65ul in end repaired step.
  *** After Nanodrop check, it was grouped into Gr.2
  *** After Nanodrop check, it was grouped into Gr.2


===Adapter ligation===
==Adapter ligation==


'''Prepare adapters'''


==== Adapter calculation ====
 
  Since A-tailed DNAs were divided into two groups baded on conc. range adapter was prepared in two different conc. according to maximum amount of A-tailed DNA in each group.
  Since A-tailed DNAs were divided into two groups baded on conc. range adapter was prepared in two different conc. according to maximum amount of A-tailed DNA in each group.


Line 88: Line 87:


  '''Gr.2'''
  '''Gr.2'''
  For 35 ng dA-tailed product (MG8) = 342ng / 303,857 g/mole =  11.2E-4 nmole * 20:1 = 0.0225 nmole adapters required.
  For 35 ng dA-tailed product (MG8) = 35ng / 303,857 g/mole =  1.1E-4 nmole * 20:1 = 0.00225 nmole adapters required.
  Adapters (ul) = 0.0225nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.0225nmoles/ (20E-3 nmoles/ul) = 1.1 ul  
  Adapters (ul) = 0.00225nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.00225nmoles/ (20E-3 nmoles/ul) = 0.11 ul  
  -----> 1.1 ul of 20uM adapters for dA-tailed products of Gr.1.
  -----> 1.1 ul of 20uM adapters for dA-tailed products of Gr.1.




  Stoffel buffer                    6ul
==== Adapter preparation ====
  PE_t_adapter                      2ul
                                                                       
  PE_b_adapter                      2ul  
  Stoffel buffer                    6ul                
  -------------------
  PE_t_adapter                      2ul                
  Total
  PE_b_adapter                      2ul                
  -------------------------------------- 
  Total                             10ul             


  94C 2min -> 0.2C/sec to 20C -> 4C hold
  94C 2min -> 0.2C/sec to 20C -> 4C hold
 
                                              '''x8.2 mix for Gr.1'''  '''x5.2 mix for Gr.2'''
  SampleID  DNA(ul)          Adapters(ul)         QuickLigase(ul)    2xQuickLigase Buffer(ul)
A-tailed DNA             35ul                        -                        -
 
Adapters                 1ul                          8.2ul                    5.2ul (Adapter was diluted 10x in Stoffel buffer)
QuickLigase               2ul                          16.4                      0.4                         
2xQuickLigase Buffer     38ul                        312                      198
--------------------------------------------------------------------------------------
Total                    76ul                  - aliquot 41ul into each tube & add 35ul of A-tailed DNA
    
    
  Incubate at RT fo 15 min. Purify with 0.7x AMPure beads. Elute with 40ul EB.
  - Incubate at RT fo 15 min. Purify with 0.7x AMPure beads. Elute with 40ul EB.


==Amplification==
==Amplification==
Line 112: Line 117:
   2.0ul  PCR_R.N2IndX(10uM) [X=1,2,3,4,...12]
   2.0ul  PCR_R.N2IndX(10uM) [X=1,2,3,4,...12]
   0.4ul  SYBR Green 50x
   0.4ul  SYBR Green 50x
  35.6ul  H2O
  30.6ul  H2O
  50.0ul  Phusion HF, 2xMM
  50.0ul  Phusion HF, 2xMM
  --------------------------------
  --------------------------------

Revision as of 15:46, 29 October 2010

Mouse gut SAGs Library Construction

(copied from Dinh's page and modified based on the actual experiment)

ID
MG1  
MG2
MG4
MG5
MG8
MG9
MG12
MG13
MG14
MG15
MG16
MG32

Sheared DNA (approx. size 300bp) amount 1ug in total volume 100ul except MG12, 5ug in 100ul


End Repair

(NEB)                                               x 13rxn mix
sheared DNA                       85ul              -
10x End Repair reaction buffer    10ul              130ul
End-repair enzyme mix             5ul               65
------------------------------------------          - aliquot 15ul of reaction mix into PCR tube
Total                             100ul             - add 85ul of sheared DNA (except MG12 add 17ul, adjust volume to 100ul with EB)
                                                    
- incubate at room temperature for 30 minutes, purify with 0.7x AMPure beads, elute with 40ul EB. 

Quick blunting Kit - repeat blunting because MDA buffer and enzyme may affect End-repair efficiency in previous step.
(NEW)                                                x 13rxn mix
DNA                                38ul              -
10X blunting bufer.                5ul               65ul              
dNTP mix                           5ul               65ul
Blunting Enzyme                    2ul               26
------------------------------------------           - aliquot 12ul of reaction mix into PCR tube,
                                                     - add 38ul of DNA 
                                                     
- incubate at room temperature for 30 minutes, purify with 0.7x AMPure beads, elute with 40ul EB.
                                

dA-tailing

(NEB)                                                x 14rxn mix
End repaired DNA                   37ul               -
dA-tailing buffer, 10x             5ul                70ul    
Klenow Fragment                    3ul                42ul
-------------------------------------------           - aliquot 13ul of reaction mix into PCR tube
Total                              50ul               - add 37ul of end repaired DNA
- incubate in thermomixer at 37C for 30 minutes, purify with 0.7x AMPure beads, elute with 40ul EB.
- quantify each reaction with Nanodrop and PAGE quantification.
ID             Conc.(ng/ul)by Nanodrop Conc.(ng/ul)by PAGE quantification
MG1                     -0.1                               1.66*                
MG2                     2.6                                8.05*
MG4                     3.2                                9.77*
MG5                     2.3                                5.04*
MG8                     0.8                                0.99**
MG9                     0.6                                3.18*
MG12                    0.2                                -
MG13                    0.3                                0.14**
MG14                   -0.5                                0.27**
MG15                   -0.0                                2.62*
MG16                   -0.2                                0.73**
MG32                    0.3                                1.35* 
NTC                     0.5                                - 
-From DNA conc. by PAGE quantification, DNA samples were divided in two subgroups 
- Gr.1 (*) Conc.>1.0ng/ul: MG1, MG2, MG4, MG5, MG9, MG15 & MG32
- Gr.2 (**) Conc.<1.0ng/ul: MG8, MG13, MG14 & MG16
- Sample MG12 didn't show the product in the gel then it was repeated again with larger amount of sheared DNA, 65ul in end repaired step.
*** After Nanodrop check, it was grouped into Gr.2

Adapter ligation

Adapter calculation

Since A-tailed DNAs were divided into two groups baded on conc. range adapter was prepared in two different conc. according to maximum amount of A-tailed DNA in each group.
Adapters to ligated product ratio: 20:1
Gr.1
Est length of dA-tailed products: 500bp (based on the sized showed in the gel)
MW dA-tailed products = (500bp*607.4 D/bp +157.9 D) = 303.857kD = 303,857 g/mole
For 342 ng dA-tailed product (MG4) = 342ng / 303,857 g/mole =  11.2E-4 nmole * 20:1 = 0.0225 nmole adapters required.
Adapters (ul) = 0.0225nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.0225nmoles/ (20E-3 nmoles/ul) = 1.1 ul 
-----> 1.1 ul of 20uM adapters for dA-tailed products of Gr.1.
Gr.2
For 35 ng dA-tailed product (MG8) = 35ng / 303,857 g/mole =  1.1E-4 nmole * 20:1 = 0.00225 nmole adapters required.
Adapters (ul) = 0.00225nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.00225nmoles/ (20E-3 nmoles/ul) = 0.11 ul 
-----> 1.1 ul of 20uM adapters for dA-tailed products of Gr.1.


Adapter preparation

Stoffel buffer                    6ul                 
PE_t_adapter                      2ul                 
PE_b_adapter                      2ul                 
--------------------------------------  
Total                             10ul               
94C 2min -> 0.2C/sec to 20C -> 4C hold
                                             x8.2 mix for Gr.1   x5.2 mix for Gr.2
A-tailed DNA              35ul                         -                         -
Adapters                  1ul                          8.2ul                     5.2ul (Adapter was diluted 10x in Stoffel buffer)
QuickLigase               2ul                          16.4                      0.4                          
2xQuickLigase Buffer      38ul                         312                       198
--------------------------------------------------------------------------------------
Total                     76ul                  - aliquot 41ul into each tube & add 35ul of A-tailed DNA
 
- Incubate at RT fo 15 min. Purify with 0.7x AMPure beads. Elute with 40ul EB.

Amplification

  1x    Reagents
15 ul  adapter ligased DNA
 2.0ul  PCR_F(10uM) 
 2.0ul  PCR_R.N2IndX(10uM) [X=1,2,3,4,...12]
 0.4ul  SYBR Green 50x
30.6ul  H2O
50.0ul  Phusion HF, 2xMM
--------------------------------
100 ul each x 3 per sample

Program

98C - 30s, (98C - 10s, 62C - 20s, 70C - 30s)x10/12, 72C - 2min.

Size selection

Select 414bp band. Or 400-425bp band.