Noi/NOTES/2013-4-16

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Low input (1ng) library preparation using Kapa kit (Continued)[edit]

  • The main point of doing this experiment is to try preparing sequencing library of Jeff's samples using ligation-based method. However, it didn't work well, and I couldn't get it work for the positive control of 1ng DNA input. There could be the limitation of DNA input and the adapter to template ratio. I would do experiment by varying amount of DNA input of 1, 2, 5 and 10ng, and also included 2 of Jeff' samples.
  • I prepared DNA for shearing using NA12878 samples
  • Confirm concentration by dsDNA Qubit Quantification (100ng/ul). By using Nanodrop the concentration is 219ng/ul
  • Dilute DNA to 1ng/ul (2ul of 100ng/ul in total volume 200ul with H2O)
  • Dilute to different concentration and aliquot 50ul into microtube
' 1ng 2ng 5ng 10ng
DNA 1ng/ul 2.20 4.40 11.00 22.00
H2O 107.80 105.60 99.00 88.00
Total amount in 50ul (ng) 1.00 2.00 5.00 10.00
  • Sheared DNA into 400bp fragments
  • Performed library prep using the same procedure as I did on April 12, 2013. Use amount of adapter calculated for 10ng DNA template (20:1) for all samples. This amount of adapters gave different ratio of adapter: template for differen input -> 200:1 for 1ng, 100:1 for 2ng, 40:1 for 5ng and 20:1 for 10ng.
  • Note: I normally used 20:1 for ligation with minimum DNA input 100ng
  • Samples: NTC, 1ng, 2ng, 5ng, 10ng, Jeff #4 and Jeff #5
  • After ligation, eluted with 20ul H2O and use all for qPCR.
Components 1x rxn 8.5 rxn mix
adapter ligated DNA 20.00 0.00
PCR_F(10uM) 1.00 8.50
PCR_R.N2IndX(10uM) 1.00 0.00
H2O 28.00 238.00
2X KAPA SYBR MM 50.00 425.00
Total volume 100.00 850.00
  • Aliquot 79ul, add 1ul PCR_R.N2.Ind, 20ul template
Program
98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x20, 72C - 2min, hold at 15C
Samples Indx
1ng PCR_R.N2Indx1
2ng PCR_R.N2Indx2
5ng PCR_R.N2Indx3
10ng PCR_R.N2Indx4
Jeff#4 PCR_R.N2Indx5
Jeff#5 PCR_R.N2Indx6
NTC2 PCR_R.N2Indx7
NTC1 PCR_R.N2Indx8
File:20130416 qPCR LowInput varyinput.png
  • Note: The qPCR curve of 2ng and 5ng were pretty much the same, but the signal of 2ng came from the adapter as the intensity of ~150bp band was stronger than the 5ng reaction.
  • PAGE analysis to verify the PCR products by loading 5ul of PCR product without bead purification
Loaded 5ul of PCR products
File:ZhangLab 2 2013-04-16 19hr 25min 5ul.jpg
Loaded 15ul of PCR products
File:ZhangLab 2 2013-04-16 20hr 17min 15ul.jpg
Note: NTC1 = NTC without adapters (I only added H2O in qPCR, 
      NTC2 = NTC with adapters (from the beginning steps)
  • From the image,I saw amplification in 5ng and 10ng, I barely saw the signal in 2ng. I loaded more of product (15ul), but the signal in 2ng was still very faint. There is no amplification in Jeff' sample and 1ng control. It clearly showed that the amount of DNA input as low as 1ng is the case that the library prep dind't work in my hands.This may be the reason that I could not prepare library using Jeff' samples which have very low amount of DNA input.
  • Need suggestion and discusion from Dr. Zhang and Jeff based on these results.

2013_04_18[edit]