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 (Note: since the beads will be reused during library prep, I didn't dilute the beads).  
For details of each step, epMotion log file will be added later). Protocol's name: XXX
* Purify sheared DNA with 1volume AmPURE beads (50ul) and elute with 50ul EB buffer and keep the bead in the well. (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)
* 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.
* Estimate concentration by PAGE quantification (to use this amount as the average 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| 400px]]
[[File:ZhangLab_2 2013-01-15 12hr 28min_purified_shearedDNA.jpg| 400px]]
{| {{table}}
{| {{table}}
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| H1||1.80||90.23
| 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.
* 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, ~100ng total.
== End repair ==
== End repair ==
{| {{table}}
{| {{table}}
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|-
|-
| End repair reaction mix ||20.00||ul
| End repair reaction mix ||20.00||ul
|}<br>
|}
* 20C for 30min
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''KAPA End repair mix'''
| align="center" style="background:#f0f0f0;"|'''KAPA End repair mix'''
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* Aliquot to column #9: 22ul
* Aliquot to column #9: 22ul
== A-tailing ==
== A-tailing ==
A-tailing reaction mix: Total 30ul/reaction<br>
* A-tailing reaction mix: Total 30ul/reaction
* 30C for 30min
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''A-tailing reaction mix'''
| align="center" style="background:#f0f0f0;"|'''A-tailing reaction mix'''
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== Adapter ligation ==
== Adapter ligation ==
* Adapter ligation mix: Total 40ul/reaction -> 10ul is adapters
* Adapter ligation mix: Total 40ul/reaction -> 10ul is adapters
* 20C for 15min<br>
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Ligation reaction mix'''
| align="center" style="background:#f0f0f0;"|'''Ligation reaction mix'''
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|}
|}
* Aliquot to column #12: 13ul
* Aliquot to column #12: 13ul
 
* After finish adapter ligation, adapter ligated DNA was resuspended in 15ul EB buffer
== Amplification ==
== Amplification ==
* Test if amplification works in small volume 25ul (add 1.25ul of adapter ligated template)
* '''TEST''' if amplification works in small volume 25ul (add 1.25ul of adapter ligated template)
{| {{table}} border = 1
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
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| PCR_F(10uM)||0.50||3.00
| PCR_F(10uM)||0.50||3.00
|-
|-
| PCR_R.N2IndX(10uM)||0.50||3.00
| PCR_R (10uM)||0.50||3.00
|-
|-
| SYBR Green 50x||0.10||0.60
| SYBR Green 50x||0.10||0.60
Line 134: Line 138:
  '''Program'''
  '''Program'''
  98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x12, 72C - 2min, hold at 15C
  98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x12, 72C - 2min, hold at 15C
* PAGE analysis (loaded PCR product 2ul)
* PAGE analysis to see the pattern of amplified amplicons (loaded PCR product 2ul, no bead purification)
[[File:ZhangLab_2 2013-01-15 21hr 12min_Test_PCR.jpg| 300px]]
[[File:ZhangLab_2 2013-01-15 21hr 12min_Test_PCR.jpg| 300px]]
* Since PCR worls well, I amplified all adapter ligated DNA (3 replicates, 5ul of template in 100ul reaction). I reduced number of cycle from 12 to 11 cycles
* Since PCR worked well, I amplified all adapter ligated DNA (B1-E1, 3 replicates, 5ul of template in 100ul reaction). I reduced number of cycle from 12 to 11 cycles
{| {{table}} border = 1
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
Line 159: Line 163:
  '''Program'''
  '''Program'''
  98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x11, 72C - 2min, hold at 15C
  98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x11, 72C - 2min, hold at 15C
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Position in<br>AC-718-384-well'''
| align="center" style="background:#f0f0f0;"|'''Position in<br> AC-718-96-well_plate1'''
| align="center" style="background:#f0f0f0;"|'''Individual library ID'''
| align="center" style="background:#f0f0f0;"|'''Indx'''
|-
| J03||B1||718_plate1_B1||PCR_R.N2Indx1
|-
| L02||C1||718_plate1_C1||PCR_R.N2Indx2
|-
| M05||D1||718_plate1_D1||PCR_R.N2Indx3
|-
| N03||E1||718_plate1_E1||PCR_R.N2Indx4
|}
* Pooled 80ul of amplified amplicons from the three replicates (total 240ul) and bead purified (diluted Ampure beads 3X with 20%PEG) with epMotion.
* Eluted with EB buffer total volme 75ul
* Will do PAGE quantification

Revision as of 15:13, 16 January 2013

Skin Microbiom Project

Library construction

For details of each step, epMotion log file will be added later). Protocol's name: XXX
  • Purify sheared DNA with 1volume AmPURE beads (50ul) and elute with 50ul EB buffer and keep the bead in the well. (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 quantification (to use this amount as the average 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, ~100ng total.

End repair

Total reaction 70.00 ul
DNA 50.00 ul
End repair reaction mix 20.00 ul
  • 20C for 30min
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
  • 30C for 30min
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
  • 20C for 15min
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.
20uM adapters (ul) required = 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
  • After finish adapter ligation, adapter ligated DNA was resuspended in 15ul EB buffer

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 (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 to see the pattern of amplified amplicons (loaded PCR product 2ul, no bead purification)

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

  • Since PCR worked well, I amplified all adapter ligated DNA (B1-E1, 3 replicates, 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
Position in
AC-718-384-well
Position in
AC-718-96-well_plate1
Individual library ID Indx
J03 B1 718_plate1_B1 PCR_R.N2Indx1
L02 C1 718_plate1_C1 PCR_R.N2Indx2
M05 D1 718_plate1_D1 PCR_R.N2Indx3
N03 E1 718_plate1_E1 PCR_R.N2Indx4
  • Pooled 80ul of amplified amplicons from the three replicates (total 240ul) and bead purified (diluted Ampure beads 3X with 20%PEG) with epMotion.
  • Eluted with EB buffer total volme 75ul
  • Will do PAGE quantification