Noi/NOTES/2013-4-24: Difference between revisions

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* The signal was lower than Rui with 15 cycles amplification. Rui's result showed stronger signal than background. I might optimize annealing temperature by using two step PCRs to minimize the background form adapters.
* The signal was lower than Rui with 15 cycles amplification. Rui's result showed stronger signal than background. I might optimize annealing temperature by using two step PCRs to minimize the background form adapters.
* The amount of unligated adapters after ligation reaction interfere qPCR curve.  
* The amount of unligated adapters after ligation reaction interfere qPCR curve.  
=== 2013_04_24 ===
* Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-4-25
* Receive 8 positive control samples using the CGI protocol
* Note from Jeff: I amplified e. coli gDNA using standard in tube MDA, with the addition of diluted dUTP at a very low concentration.  I then purified, and diluted the amplicons to 1 ng and 10 ng.  I then used USER to remove the dUTPs incorporated during MDA, and used Exo - klenow for nick translation and A tailing.
'''Sample list (4ul each)'''
2 10 ng, high USER conc.
2 10 ng, low USER conc.
2 1 ng, high USER conc.
2 1 ng, low USER conc.
 
* To process these samples
** Do adapter ligation, bead purification, and PCR
** Require at least 10X dilution of original volume (4ul --> need minimum ligation reaction 40ul)
** If the reaction works, we expected to see a smear from ~500 -1500bp
** Since DNA input is in the range of 1-10ng, I will used the same condition I tried on 2013_04_23 but using 50ul ligation reaction
'''Order of the tubes (need to check more info with Jeff)'''
1. 1ng high/low? USER short incubation
2. 1ng high/low? USER short incubation
3. 10ng high/low? USER short incubation
4. 10ng high/low? USER short incubation
5. 1ng high/low? USER long incubation
6. 1ng high/low? USER long incubation
7. 10ng high/low? USER long incubation
8. 10ng high/low? USER long incubation
9. NTC
== Adapter ligation ==
* Adapter ligation mix (Total 50ul) : Ligation reaction mix 49.2ul/reaction & 0.8ul of 3uM adapters (Diluted 30uM adapters 10X with cold 1X Stoffel buffer)
* I added 20ul to 4ul samples before transferring to new clear tube
{| class="wikitable" style="text-align:center;
| align="center" style="background:#f0f0f0;"|'''Ligation reaction mix'''
| align="center" style="background:#f0f0f0;"|'''1x rxn'''
| align="center" style="background:#f0f0f0;"|'''9x rxn mix'''
|-
| KAPA 5X Ligation buffer||10.00||90.00
|-
| KAPA DNA ligase||5.00||45.00
|-
| H2O||10.20||91.80
|-
| Total||25.20||226.80
|}
* Add 25.2ul of Ligation reaction mix to 24ul DNA samples
* Add 0.8ul of 3uM adapters
* 20C for 15min (no shaking)
* Bead purification: 50ul 20% PEG/5M NaCl, wait 10min (no shaking), sit on magnet 5min, wash twice with fresh 80% EtOH, dry for 3 min
* Eluted with 15ul H2O (used all for PCR)
== Amplification ==
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1x rxn'''
| align="center" style="background:#f0f0f0;"|'''9.5 rxn mix'''
|-
| adapter ligated DNA||15.00||0.00
|-
| PCR_F(10uM)||1.00||9.50
|-
| PCR_R.N2IndX(10uM)||1.00||0.00
|-
| H2O||8.00||76.00
|-
| 2X KAPA SYBR MM||25.00||237.50
|-
| Total volume||50.00||475.00
|}
* Aliquot 34ul, add 1ul of PCR_R.N2Indx, 15ul of adapter ligated DNA
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Samples'''
| align="center" style="background:#f0f0f0;"|'''Indx'''
|-
| 1. 1ng high/low? USER _S||PCR_R.N2Indx1
|-
| 2. 1ng high/low? USER _S||PCR_R.N2Indx2
|-
| 3. 10ng high/low? USER _S||PCR_R.N2Indx3
|-
| 4. 10ng high/low? USER _S||PCR_R.N2Indx4
|-
| 5. 1ng high/low? USER _L||PCR_R.N2Indx5
|-
| 6. 1ng high/low? USER _L||PCR_R.N2Indx6
|-
| 7. 10ng high/low? USER _L||PCR_R.N2Indx7
|-
| 8. 10ng high/low? USER _L||PCR_R.N2Indx8
|-
| 9. NTC||PCR_R.N2Indx8
|}
'''Program (Eppendorf Realplex)'''
98C - 30s, (98C - 10s, 62C - 30s, 72C - 30s)x20, 72C - 2min, hold at 15C
* Jeff suggested not go over 15 cycles. I stopped reaction at 15 cycles.
* Will add qPCR curve and gel image
* I didn't see amplification in all samples. NTC showed stronger signal from adapter than all samples. Did some components from CGI protocol interfere PCR since I expected to see the same adapter background if the ligation did not work? Why the background in all samples was consistently low? Does 30 sec for extension too short to amplify ~500-1500bp. I forgot to think about this. Next time, I should increase more extension time to 1min
* Jeff found out that dNTPs were not added during nick translation step, so I will repeat experiment using the same condition with minor adjustment of extension time.

Revision as of 01:57, 26 April 2013

Low input (1ng) library preparation using Kapa kit (updated)

2013_04_23

  • Even Jeff will process his MDA products using CGI protocol, we still want to try the standard protocol following Rui's condition she used for her experiment.
  • Things have been adjusted
    • DNA shearing: I included the 200bp fragment to compare if the size effects adapter ligation efficiency (Rui: 200bp fragment)
    • Prepared the same concentration of adapter (30uM) and used the same concentration for ligation
    • Amplification:
      • Annealing temperature: I previously used 65C for annealing. I reduced annealing temperature to 62C (Rui: 60C with the same pair of primers).
      • Reduced volume from 100ul to 50ul
      • Slightly changes were noted during each step
  • Sample list: I still included higher amount of DNA (2.5 & 5ng) to compare the improvement by adjusting the conditions.
    • NTC, 1ng_200bp, 2.5ng_200bp, 5ng_200bp, 1ng_400bp, 2.5ng_400bp, 5ng_400bp

End repair

Total reaction 70.00 ul
DNA 50.00 ul
End repair reaction mix 20.00 ul
KAPA End repair mix 1x rxn 7x rxn mix
10X End repai buffer 7.00 49.00
KAPA End repair enzyme 3.50 24.50
H2O 9.50 66.50
Total 20.00 140.00
  • 20C for 30min (no shaking)
  • Bead purification: 112ul AMPure beads, wait 10min (no shaking), sit on magnet 5min, wash twice with fresh 80% EtOH, dry for 3 min
  • Add 20ul of H2O to resuspend the beads first to make sure that all DNAs were completely dissolved (keep the beads) before adding A-tailing reaction mix

A-tailing

  • A-tailing reaction mix: Total 50ul/reaction (increase from 30ul to 50ul)
  • DNAs were dissolved with 20ul H2O in the previous step (the beads were carried over till the last step)
KAPA End repair mix 1x rxn 7x rxn mix
10X End repair buffer 7.00 49.00
KAPA End repair enzyme 3.50 24.50
H2O 9.50 66.50
Total 20.00 140.00
  • Add 30ul of A-tailing reaction mix to 20ul dissolved DNA
  • 30C for 30min (no shaking)
  • Bead purification: 90ul 20% PEG/5M NaCl, wait 10min (no shaking), sit on magnet 5min, wash twice with fresh 80% EtOH, dry for 3 min
  • Add 20ul of H2O to resuspend the beads as described in A-tailing (keep the beads) before adding ligation reaction mix and adapters

Adapter ligation

  • Adapter ligation mix (Total 40ul) : Ligation reaction mix 39.2ul/reaction & 0.8ul of 3uM adapters (Diluted 30uM adapters 10X with cold 1X Stoffel buffer)
  • DNAs were dissolved with 20ul H2O in the previous step
Ligation reaction mix 1x rxn 7x rxn mix
KAPA 5X Ligation buffer 8.00 56.00
KAPA DNA ligase 4.00 28.00
H2O 7.20 50.40
Total 19.20 134.40
  • Add 19.2ul of Ligation reaction mix to 20ul dissolved DNA
  • Add 0.8ul of 3uM adapters
  • 20C for 15min (no shaking)
  • Bead purification: 50ul 20% PEG/5M NaCl, wait 10min (no shaking), sit on magnet 5min, wash twice with fresh 80% EtOH, dry for 3 min
  • Eluted with 15ul H2O (used all for PCR)

Amplification

Components 1x rxn 8.5 rxn mix
adapter ligated DNA 15.00 0.00
PCR_F(10uM) 1.00 8.50
PCR_R.N2IndX(10uM) 1.00 0.00
H2O 8.00 68.00
2X KAPA SYBR MM 25.00 212.50
Total volume 50.00 425.00
  • Aliquot 34ul, add 1ul of PCR_R.N2Indx, 15ul of adapter ligated DNA
Samples Indx
1ng_200bp PCR_R.N2Indx1
2.5ng_200bp PCR_R.N2Indx2
5ng_200bp PCR_R.N2Indx3
1ng_400bp PCR_R.N2Indx4
2.5ng_400bp PCR_R.N2Indx5
5ng_400bp PCR_R.N2Indx6
NTC1 with adapters PCR_R.N2Indx7
NTC2 no adapters PCR_R.N2Indx8
Program (Eppendorf Realplex)
98C - 30s, (98C - 10s, 62C - 30s, 72C - 30s)x20, 72C - 2min, hold at 15C
  • I set up total 20 cycles, but monitored to stop at any number of cycle before reaching a plateau
  • Stop at 13 cycles
qPCR curve
File:20130424 qPCR LowInpu.png
  • PAGE analysis to verify the PCR products by loading 5 ul of PCR product without bead purification (1/10 of total volume)
File:20130424 qPCR LowInput.png
  • From the qPCR curve and gel image, the background from adapters was very high comparing to the signal of expected bands. The qPCR curves between NTC and experiment were very close for very low input. Verification by PAGE telling more information
  • 2.5ng and 5ng clearly showed amplification. The background should be clean after bead purification. Still did not see amplification for 1ng sample either 200bp or 400bp
  • These conditions show more improvement comparing to previous experiment with 20 cycles.
  • No significant different between 200bp and 400bp DNA fragments. Seem like 400bp show slightly stronger signal.
  • The signal was lower than Rui with 15 cycles amplification. Rui's result showed stronger signal than background. I might optimize annealing temperature by using two step PCRs to minimize the background form adapters.
  • The amount of unligated adapters after ligation reaction interfere qPCR curve.
  • Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-4-25