Noi/NOTES/2013-3-12: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Noi
mNo edit summary
>Noi
mNo edit summary
Line 103: Line 103:
| Total||||||||||30.00
| Total||||||||||30.00
|}
|}
== Amplification TEST ==
* There are two set of primers
* '''Set 1''': 1 forward primer without barcode && 3 different reverse primers with different barcodes (Indx1-3)
{| {{table}}
| 1-1. gDNA (12878)||Indx1
|-
| 2-1. Total RNA||Indx2
|-
| 3-1. ds cDNA no RNasA treatment||Indx3
|-
| 4-1. ds cDNA RNaseA treatment||Indx3
|}
* '''Set 2''': 3 different forward primers with different barcodes  &&  combination of 9 reverse primer without barcodes
{| {{table}}
| 1-2. gDNA (12878)||Indx1
|-
| 2-2. Total RNA||Indx2
|-
| 3-2. ds cDNA no RNasA treatment||Indx3
|-
| 4-2. ds cDNA RNaseA treatment||Indx3
|}
* Note: Since we have only 3 different indexes, and the two set of indexes are the same,  the libraries amplified by the two sets of primers can not be pooled together and sample 3-1 and 3-2 will not combinded in the pooled
=== PCR TEST set up ===
=== Set 1: by Noi ===
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Sample set 1'''
| align="center" style="background:#f0f0f0;"|'''Index'''
| align="center" style="background:#f0f0f0;"|'''Primer names xxxxxxxxxxxxxxxxx'''
|-
| 1-1. gDNA (12878)||Indx1||
|-
| 2-1. Total RNA||Indx2||
|-
| 3-1. ds cDNA no RNasA treatment||Indx3||
|-
| 4-1. ds cDNA RNaseA treatment||Indx3||
|}
'''PCR Mix'''<br>
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1 rxn'''
| align="center" style="background:#f0f0f0;"|'''5.5 rxn mix'''
|-
| Captured template||1.00||0.00
|-
| 10uM Forward no barcodes||0.40||2.20
|-
| 10uM Reverse +Indx||0.40||0.00
|-
| 2x KAPA SYBG fast MM||12.50||68.75
|-
| H2O||10.70||58.85
|-
| Total||25.00||137.50
|}
* Aliqout 23.6ul, add 0.4ul of reverese primer, add 1ul of circular DNA
=== Set 2 : by Matt ===
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Sample set 2'''
| align="center" style="background:#f0f0f0;"|'''Index'''
| align="center" style="background:#f0f0f0;"|'''Primer names xxxxxxxxxxxxxxxxx'''
|-
| 1-2. gDNA (12878)||Indx1||
|-
| 2-2. Total RNA||Indx2||
|-
| 3-2. ds cDNA no RNasA treatment||Indx3||
|-
| 4-2. ds cDNA RNaseA treatment||Indx3||
|}
'''PCR Mix'''<br>
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1 rxn'''
| align="center" style="background:#f0f0f0;"|'''5.5 rxn mix'''
|-
| Captured template||1.00||0.00
|-
| 10uM Forward + Indx||0.40||0.00
|-
| 10uM Reverse no barcodes||0.40||2.20
|-
| 2x KAPA SYBG fast MM||12.50||68.75
|-
| H2O||10.70||58.85
|-
| Total||25.00||137.50
|}
* '''Program'''
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min<br>
* Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-14

Revision as of 20:14, 15 March 2013

Capture set up (Matt's project)

  • Set up capture in 30ul
NTC (no targets)
1-1. gDNA (12878)
2-1. Total RNA
3-1. ds cDNA no RNasA treatment
4-1. ds cDNA RNaseA treatment
1-2. gDNA (12878)
2-2. Total RNA
3-2. ds cDNA no RNasA treatment
4-2. ds cDNA RNaseA treatment

Probe calculation

Probe:target 1000:1 '
Probe size 12,335 probes
Template 300 ng
Human gDNA MW 1.9500E+12 g/mol (3E+09 * 650Da/bp+157.9Da)
Human gDNA (300ng) 1.5385E-19 mol (300ng/1.9500E+12 g/mol)
Probe required (1000:1) 1.5385E-16 mol (1.5385E-19mol x 1000)
Probe MW (12,335 probes, 170nt) 6.8151E+08 g/mol (12335 probes * 170nt * 325Da/nt + 79Da)
Amount probe required 1.05E-07 g (6.8151E+08 g/mol * 1.5385E-16mol)
Amount probe required 105.00 ng
  • Since I resuspend the probes in too large volume, I concentrate using Qiaquick column, elute in total volume ~62ul, re-quantify conc. ~22ng/ul
  • Note:
    • I showed Matt how to calculate probes and he did calculation, and the number he got agreed with this calculation
    • Even the target amounts in each reaction are not the same, I used the same probe amount in every reaction based on gDNA calculation

Probes and AmpLigase Buffer Mix

Components Volume (ul) 9.5 rxn mix
12K Insitu probes (22ng/ul) 4.77 45.32
10X AmpLigase buffer 3.00 28.50
Total 7.77 73.82
Samples 12K Insitu probes 10X AmpLigase buffer Conc. (ng/ul) Targets (ul) H2O (ul) Total (ul) Total amount
in capture (ng)
NTC 4.77 3.00 0 0 22.23 30.00 0.00
1-1. gDNA (12878) 4.77 3.00 45.10 6.65 15.58 30.00 299.92
2-1. Total RNA 4.77 3.00 1000.00 1.00 21.23 30.00 1000.00
3-1. ds cDNA no RNasA treatment 4.77 3.00 15.20 19.00 3.23 30.00 288.80
4-1. ds cDNA RNaseA treatment 4.77 3.00 5.84 19.00 3.23 30.00 110.96
1-2. gDNA (12878) 4.77 3.00 45.10 6.65 15.58 30.00 299.92
2-2. Total RNA 4.77 3.00 1000.00 1.00 21.23 30.00 1000.00
3-2. ds cDNA no RNasA treatment 4.77 3.00 10.20 19.00 3.23 30.00 193.80
4-2. ds cDNA RNaseA treatment 4.77 3.00 7.77 19.00 3.23 30.00 147.63

Program

  • 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
  • -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
  • -> 55 C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

AmpLigase enzyme mix

Components Stock conc. Unit Final conc. Unit Prepare volume 30ul
AmpLigase 5 U/ul 0.5 U/ul 3.00
10x AmpLigase Buffer 10 x 1 x 3.00
H2O 24.00
Total 30.00