Noi/NOTES/2013-1-15: Difference between revisions

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= Skin Microbiom Project =
= Skin Microbiom Project =
= Library construction =
= Library construction =
* Purify sheared DNA with 1volume AmPURE beads (50ul) and elute with 50ul EB buffer.  
* Purify sheared DNA with 1volume AmPURE beads (50ul) and elute with 50ul EB buffer (Note: since the beads will be reused during library prep, I didn't dilute the beads).  
* Note: Lost sample in well A1 (well broken)
* Note: Lost sample in well A1 (well broken)
* Dr. Zhang suggested to prepare 7 libraries, but keep 3 of them (F1-H1) to be used later and do PCR on 4 samples (B1-E1)
* Dr. Zhang suggested to prepare 7 libraries, but keep 3 of them (F1-H1) to be used later and do PCR on 4 samples (B1-E1)

Revision as of 06:42, 16 January 2013

Skin Microbiom Project

Library construction

  • Purify sheared DNA with 1volume AmPURE beads (50ul) and elute with 50ul EB buffer (Note: since the beads will be reused during library prep, I didn't dilute the beads).
  • Note: Lost sample in well A1 (well broken)
  • Dr. Zhang suggested to prepare 7 libraries, but keep 3 of them (F1-H1) to be used later and do PCR on 4 samples (B1-E1)
  • Estimate concentration by PAGE analysis (to use this amount as the roughly amount of the future experiment by assume that each sample has equal amount of DNA.

File:ZhangLab 2 2013-01-15 12hr 28min purified shearedDNA.jpg

WellID Conc. (ng/ul) Yields in 50ul
B1 0.26 13.21
C1 1.60 79.87
D1 3.92 196.11
E1 2.57 128.49
F1 4.67 233.58
G1 1.56 78.03
H1 1.80 90.23
  • From the concentration above, the total amount of amlpicons is not as high as 1ug, but in the range ~20-230ng. Taking the average of the total amount of all 7 samples, it would it ~100ng total.

End repair

Total reaction 70.00 ul
DNA 50.00 ul
End repair reaction mix 20.00 ul


KAPA End repair mix 1x rxn 8x rxn mix
10X End repai buffer 7.00 56.00
KAPA End repair enzyme 5.00 40.00
H2O 8.00 64.00
Total 20.00 160.00
  • Aliquot to column #9: 22ul

A-tailing

A-tailing reaction mix: Total 30ul/reaction

A-tailing reaction mix 1x rxn 8x rxn mix
KAPA 10X A-tailing buffer 3.00 24.00
KAPA A-tailing enzyme 3.00 24.00
H2O 24.00 192.00
Total 30.00 240.00
  • Aliquot to column #10: 32ul

Adapter ligation

  • Adapter ligation mix: Total 40ul/reaction -> 10ul is adapters
Ligation reaction mix 1x rxn 8x rxn mix
KAPA 5 Ligation buffer 10.00 80.00
KAPA DNA ligase 4.00 32.00
H2O 26.00 208.00
Total 40.00 320.00
  • Aliquot to column #11: 43ul

Adapter preparation

Assume DNA amount ~100ng, size 400bp
MW dA-tailed products = (400bp*607.4 D/bp +157.9 D) = 243.12kD = 243,118 g/mole
For 100 ng dA-tailed product = 100ng/243118 g/mole = 4.11E-04 nmole * 20:1 = 0.0082 nmole adapters required.
Adapters (ul) = 0.0082nmoles/(20xE3 nmoles/L * 1E-06L/ul) = 0.0082nmoles/(20E-3 nmoles/ul) =  0.411ul
I will prepare total volume of  20uM annealed adapters 10ul 
Stoffel buffer 1.00
PE_t_adapter 2.00
PE_b_adapter 2.00
H2O 5.00
  • 94C 2min -> 0.2C/sec to 20C -> 4C hold
  • Dilute annealed adapter with 1x Stoffel buffer 0.41ul to 9.59ul -> anneal adapterr 4.1ul: 1x Stoffelul 95.9
20uM annealed adapter 4.10
10x Stoffel buffer 10.00
H2O 85.90
  • Aliquot to column #12: 13ul

Amplification

  • Test if amplification works in small volume 25ul (add 1.25ul of adapter ligated template)
Components 1x rxn 6x rxn mix
adapter ligated DNA 1.25 0.00
PCR_F(10uM) 0.50 3.00
PCR_R.N2IndX(10uM) 0.50 3.00
SYBR Green 50x 0.10 0.60
H2O 10.15 60.90
Phusion HF, 2xMM 12.50 75.00
Total volume 25.00 150.00
  • Aliquot 23.75ul, add 1.25 of adapter ligated DNA
Program
98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x12, 72C - 2min, hold at 15C
  • PAGE analysis (loaded PCR product 2ul)

File:ZhangLab 2 2013-01-15 21hr 12min Test PCR.jpg

  • Since PCR worls well, I amplified all adapter ligated DNA (3 replicated, 5ul of template in 100ul reaction). I reduced number of cycle from 12 to 11 cycles
Components 1x rxn 13.5rxn mix
adapter ligated DNA 5.00 0.00
PCR_F(10uM) 2.00 27.00
PCR_R.N2IndX(10uM)[X=1-4] 2.00 0.00
SYBR Green 50x 0.80 10.80
H2O 40.20 542.70
Phusion HF, 2xMM 50.00 675.00
Total volume 100.00 1350.00
  • Aliquot 93ul, add 5 of adapter ligated DNA and 2ul of PCR_R.N2Indx
Program
98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x11, 72C - 2min, hold at 15C