Noi/NOTES/2013-3-14: Difference between revisions

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* Aliquot 86ul, add 2ul of Forward Ind, 12ul of circular DNA
* Aliquot 86ul, add 2ul of Forward Ind, 12ul of circular DNA
* '''Program'''
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min<br>
* Perform bead purification , set 1 elute total volume 60ul, set 2 elute total volume 50ul.
* Verify by PAGE an do PAGE quantification to see the ratio of each product before pooling the libraries.
* Note: I quantified based on the expected band ~400bp. However the intensity of the ~250bp band was very strong I also quantified and compared the intensities to the 400bp band.
[[File:ZhangLab_2 2013-03-15 11hr 27min_SeqLib_Insitu.jpg| 400px]]
[[File:ZhangLab_2 2013-03-15 11hr 27min_SeqLib_Insitu.jpg| 400px]]
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Samples'''
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Yields (ng)'''
| align="center" style="background:#f0f0f0;"|'''Pooled volume (ul)'''
| align="center" style="background:#f0f0f0;"|'''250bp/400bp (folds)'''
|-
| gDNA-set1 400bp||7.34||440.57||40.56||2.51
|-
| dsDNA -RNaseA set1 400bp||9.93||595.71||30.00||2.37
|-
| dsDNA +RNaseA set1 400bp||9.99||599.24||30.00||2.27
|-
| gDNA-set2 400bp||6.11||305.61||3.45||3.32
|-
| dsDNA -RNaseA set2 400bp||0.53||26.39||40.00||11.03
|-
| dsDNA +RNaseA set2 400bp||1.24||61.96||17.04||6.81
|-
| gDNA-set1 250bp||18.45||||||
|-
| dsDNA -RNaseA set1 250bp||23.51||||||
|-
| dsDNA +RNaseA set1 250bp||22.63||||||
|-
| gDNA-set2 250bp||20.29||||||
|-
| dsDNA -RNaseA set2 250bp||5.82||||||
|-
| dsDNA +RNaseA set2 250bp||8.44||||||
|}

Revision as of 19:08, 15 March 2013

Set 1

Components 1rxn 6.5 rxn
Captured template 12.00 0.00
10uM Forward no barcodes 2.00 13.00
10uM Reverse +Indx 2.00 0.00
2x KAPA SYBG fast MM 50.00 325.00
H2O 34.00 221.00
Total 100.00 650.00
  • Aliquot 86ul, add 2ul of reverse Ind, 12ul of circular DNA

Set 2

Components ' '
Captured template 1rxn 6.5 rxn
10uM Forward + Indx 12.00 0.00
100uM Reverse no barcodes 2.00 0.00
2x KAPA SYBG fast MM 2.00 13.00
H2O 50.00 325.00
Total 34.00 221.00
100.00 650.00
  • Aliquot 86ul, add 2ul of Forward Ind, 12ul of circular DNA
  • Program

98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min

  • Perform bead purification , set 1 elute total volume 60ul, set 2 elute total volume 50ul.
  • Verify by PAGE an do PAGE quantification to see the ratio of each product before pooling the libraries.
  • Note: I quantified based on the expected band ~400bp. However the intensity of the ~250bp band was very strong I also quantified and compared the intensities to the 400bp band.

File:ZhangLab 2 2013-03-15 11hr 27min SeqLib Insitu.jpg

Samples Conc. (ng/ul) Yields (ng) Pooled volume (ul) 250bp/400bp (folds)
gDNA-set1 400bp 7.34 440.57 40.56 2.51
dsDNA -RNaseA set1 400bp 9.93 595.71 30.00 2.37
dsDNA +RNaseA set1 400bp 9.99 599.24 30.00 2.27
gDNA-set2 400bp 6.11 305.61 3.45 3.32
dsDNA -RNaseA set2 400bp 0.53 26.39 40.00 11.03
dsDNA +RNaseA set2 400bp 1.24 61.96 17.04 6.81
gDNA-set1 250bp 18.45
dsDNA -RNaseA set1 250bp 23.51
dsDNA +RNaseA set1 250bp 22.63
gDNA-set2 250bp 20.29
dsDNA -RNaseA set2 250bp 5.82
dsDNA +RNaseA set2 250bp 8.44