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

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==Amplification==
==Amplification==
  1x    Reagents
                                                              '''x28 mix'''   
15 ul adapter ligased DNA
  adapter ligased DNA                     15 ul                    -
  2.0ul PCR_F(10uM)  
  PCR_F(10uM)                             2.0ul                   56ul
  2.0ul  PCR_R.N2IndX(10uM) [X=1,2,3,4,...12]
  PCR_R.N2IndX(10uM) [X=1,2,3,4,...12]     2.0ul                    -
  0.4ul SYBR Green 50x
  SYBR Green 50x                           0.4ul                    11.2ul
  30.6ul H2O
  H2O                                      30.6ul                   857ul
50.0ul Phusion HF, 2xMM
  Phusion HF, 2xMM                         50.0ul                  1400ul
  --------------------------------
  ---------------------------------------------------------------------------
  100 ul each x 3 per sample
  Total                                    100ul                    - aliquot 83ul of rxn mix to each tube then add 15ul of adapter ligated DNA
                                          x2 for each sample      and 2ul of PCR_R.N2IndX [X=1,2,3,4....,...12]


'''Program'''
'''Program'''
  98C - 30s, (98C - 10s, 62C - 20s, 70C - 30s)x10/12, 72C - 2min.
  98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x12, 72C - 2min, hold at 15C


===Size selection===
===Size selection===
Select 414bp band.
Select 414bp band.
Or 400-425bp band.
Or 400-425bp band.

Revision as of 15:54, 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

                                                              x28 mix     
adapter ligased DNA                      15 ul                    -
PCR_F(10uM)                              2.0ul                    56ul
PCR_R.N2IndX(10uM) [X=1,2,3,4,...12]     2.0ul                    -
SYBR Green 50x                           0.4ul                    11.2ul
H2O                                      30.6ul                   857ul
Phusion HF, 2xMM                         50.0ul                   1400ul
---------------------------------------------------------------------------
Total                                    100ul                    - aliquot 83ul of rxn mix to each tube then add 15ul of adapter ligated DNA
                                         x2 for each sample       and 2ul of PCR_R.N2IndX [X=1,2,3,4....,...12]

Program

98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x12, 72C - 2min, hold at 15C

Size selection

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