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

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(copied template from Dinh's page and modified based on the actual experiment)
(copied template from Dinh's page and modified based on the actual experiment)
'''Batch ID: A'''<br><br><br>
  '''Sample ID'''
  '''Sample ID'''
   MG1   
   MG1   

Revision as of 15:56, 8 November 2010

Note: sorry, I forgot to label the images, and I'll note the image descriptions below the pictures soon.


Mouse gut SAGs Library Construction

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

Batch ID: A


Sample 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
H2O                                5ul                70ul
-------------------------------------------           - 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.

Image: 500 pixels Image: 220 pixels

Figure1. LM ladder 6ul, LM ladder 3ul, MG1, MG2, MG4, MG5, MG8, MG9, MG12, MG13, MG14, MG15
Figure2. LM ladder 6ul, LM ladder 3ul, MG16, MG32, NTC

Sample 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 based on conc. Adapter was prepared in two different conc. according to the 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
- 2ul of amplified products were randomly analyzed in PAGE, and the rest were purified by AMPure, eluted with 40ul EB.
- 2ul of AMPure purified products were analyzed again to quantify DNA conc. in the region approx. 400-600 bp.

Image: 350 pixels

Figure3. 25bp ladder, MG1, MG5, MG9, MG13, MG14, MG12, MG15, MG32, NTC


Image: 450 pixelsImage: 300 pixels

Figure4. LM 6ul, LM 3ul, MG1, MG2 MG4, MG5, MG8, MG9, MG12, MG13 Figure5. LM6ul, LM 3ul, MG14, MG15, MG16, MG32


IndX           Sample ID           Conc. ng/ul      Vol. ul (50ng)
Ind1               MG1               18.16              2.8        
Ind2               MG2               15.82              3.2
Ind3               MG4               16.21              3.1
Ind4               MG5               20.41              2.5
Ind5               MG8               21.16              2.4
Ind6               MG9               14.40              3.5
Ind7               MG12              2.38               21.0
Ind8               MG13              16.64              3.0
Ind9               MG14              21.73              2.3
Ind10              MG15              10.49              4.8
Ind11              MG16              30.77              1.6
Ind12              MG32              12.64              4.0
                                                        - Combind 50ng of each sample


Size selection

- Select 450bp band or 400 - 600bp band.
- Perform PAGE size selection with 6% TBE 2D gel by cutting the band in the range of 400 - 600bp (x4 tubes, dissolved with 11ul EB, total vol. 44ul ).

Image: 300 pixels
Figure6, MG SAGs BatchA Sequencing Library PAGE size selection (25bp ladder)


- PAGE analysis to check the size and conc. of DNA library.


- From the gel, the size of DNA range was too big (approx. 400 almost 800bp). The reason was I used 25 bp as the ladder, so I's hard to identify the
band above 500bp (couldn't find 100bp ladder).
-Dr. Zhang recommended Dinh to cut DNA in the range of 450 - 550 bp.
- Perform PAGE size selection again with 6% TBE 5-well gel, eluted with 12ul EB.
Image: 300 pixels
Figure7. MG SAGs BatchA Sequencing Library PAGE size selection (round2) 25bp ladder, 1kb ladder, 1st round PAGE SS purified sequencing library, 50bp ladder
- 1ul of DNA was analyzed in PAGE and quantify DNA conc. = 13.8ng/ul or nM

Image: 250 pixels
Figure8. LM 6ul, LM3ul, 2 round PAGE SS purified sequencing library, 1kb ladder, 25bp ladder