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

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==Amplification==
==Amplification==
   1x    Reagents
   1x    Reagents
  10.0ul adapter ligased DNA
  15 ul adapter ligased DNA
   2.0ul  PCR_F(10uM)  
   2.0ul  PCR_F(10uM)  
   2.0ul  PCR_R.N2IndX(10uM) [X=1,2,3,4,...12]
   2.0ul  PCR_R.N2IndX(10uM) [X=1,2,3,4,...12]

Revision as of 07:57, 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.
                                                     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

                                                     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.

Adapter ligation

Prepare adapters
Stoffel buffer                    6ul
PE_t_adapter                      2ul
PE_b_adapter                      2ul 
-------------------
Total
94C 2min -> 0.2C/sec to 20C -> 4C hold
Adapters to ligated product ratio: 20:1
Est length of dA-tailed products: 300bp (after shearing)
MW dA-tailed products = (300bp*607.4 D/bp +157.9 D) = 182.378kD = 182,378 g/mole
For 500 ng dA-tailed product = 500ng / 182,378g/mole =  27.4E-4 nmole * 20:1 = 0.055 nmole adapters required.
Adapters (ul) = 0.055nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.055nmoles/ (20E-3 nmoles/ul) = 2.74 ul 
-----> 2.74 ul of 20uM adapters per 500ng dA-tailed products. 
 SampleID   DNA(ul)          Adapters(ul)          QuickLigase(ul)     2xQuickLigase Buffer(ul)


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
35.6ul  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.