Noi/NOTES/2014-11-25: Difference between revisions
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= Comparison of the efficiency between TruSeq adaptor and | = Comparison of the efficiency between TruSeq adaptor and a customized unmethylated Y-adaptor with UMI = | ||
[[noi:DMR220k_LabNotes#2014|'''Back to calendar''']] | [[noi:DMR220k_LabNotes#2014|'''Back to calendar''']] | ||
* Dr. Zhang wanted to compare the efficiency of standard adaptor and a customized Y-adaptor with UMI for generating sequencing libraries using KAPA Hyper Prep kit. In this experiment, I used TruSeq adaptor to represent standard adaptor. | * Dr. Zhang wanted to compare the efficiency of standard adaptor and a customized Y-adaptor with UMI for generating sequencing libraries using KAPA Hyper Prep kit. In this experiment, I used TruSeq adaptor to represent standard adaptor. | ||
* In addition to compare between the two versions of adaptor, I also compare between a half reaction volume (30ul of end-repair/A-tailing and 55ul of ligation reaction) and a full reaction (60ul of end-repair/A-tailing and 110ul of ligation reaction) following the reaction set up of the instruction from KAPA. The reason is that the reaction is optimum for DNA input between 1ng-1ug. My experiment uses 1ng of input, so I think that the enzymes/and reagents should be more than enough. I therefore set up a half reaction volume to save the reagents and the concentrations of all components are still the same. | |||
* I received top and bottom oligos to generated Y-adaptor oligo --> final conc. 15uM to be equivalent to the concentration of TruSeq adaptor | * I received top and bottom oligos to generated Y-adaptor oligo --> final conc. 15uM to be equivalent to the concentration of TruSeq adaptor | ||
<br> | <br> | ||
'''<u>Annealing of Y-adaptor</u>''' | |||
'''Component Volume (ul)''' | |||
SG_Adt.top_v1 (100uM) 30.00 | |||
SG_Adt.bottom_v3 (100uM) 30.00 | |||
10X Stoffel Buffer 20.00 | |||
H2O 120.00 | |||
'''Total 200.00''' | |||
I split 100ul to 2X of 0.2mL PCR tube and incubate at the following templarature | |||
95C, 5min --> -0.1C/s to 20C --> hols at 20C, 5min and put on ice | |||
'''Sample list'''<br> | |||
'''No Sample IDs Conc. (ng/ul)''' | |||
#1 6P-2 1.10 | |||
#2 6P-3 1.57 | |||
#3 7P-2 1.14 | |||
* Note: I will reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally. | |||
* I set up one reaction of sample 6P-2 in a full reaction volume for comparison | |||
* KAPA Hyper Prep reaction set up (exactly in the instruction): | |||
:- 50ul of fragmented DNA | |||
:- 7ul of End repair & dA-tailing buffer | |||
:- 3ul of End repair & dA-tailing enzyme mix | |||
:- Total reaction: 60ul | |||
== End repair & dA-tailing == | |||
'''Sample IDs Adaptor Reaction volume Tube #''' | |||
6P-2 TruS half rxn #1 | |||
6P-3 TruS half rxn #2 | |||
7P-2 TruS half rxn #3 | |||
NTC TruS half rxn #4 | |||
6P-2 UMI half rxn #5 | |||
6P-3 UMI half rxn #6 | |||
7P-2 UMI half rxn #7 | |||
NTC UMI half rxn #8 | |||
7P-2 TruS full rxn #9 | |||
TruS = TruSeq adaptor | |||
UMI = customized unmethylated Y-adaptor with UMI | |||
Tube #1 and #9 will be compared the effect of total volume. | |||
==== End repair & dA-tailing reaction mix ==== |
Revision as of 01:00, 28 November 2014
Comparison of the efficiency between TruSeq adaptor and a customized unmethylated Y-adaptor with UMI
- Dr. Zhang wanted to compare the efficiency of standard adaptor and a customized Y-adaptor with UMI for generating sequencing libraries using KAPA Hyper Prep kit. In this experiment, I used TruSeq adaptor to represent standard adaptor.
- In addition to compare between the two versions of adaptor, I also compare between a half reaction volume (30ul of end-repair/A-tailing and 55ul of ligation reaction) and a full reaction (60ul of end-repair/A-tailing and 110ul of ligation reaction) following the reaction set up of the instruction from KAPA. The reason is that the reaction is optimum for DNA input between 1ng-1ug. My experiment uses 1ng of input, so I think that the enzymes/and reagents should be more than enough. I therefore set up a half reaction volume to save the reagents and the concentrations of all components are still the same.
- I received top and bottom oligos to generated Y-adaptor oligo --> final conc. 15uM to be equivalent to the concentration of TruSeq adaptor
Annealing of Y-adaptor Component Volume (ul) SG_Adt.top_v1 (100uM) 30.00 SG_Adt.bottom_v3 (100uM) 30.00 10X Stoffel Buffer 20.00 H2O 120.00 Total 200.00 I split 100ul to 2X of 0.2mL PCR tube and incubate at the following templarature 95C, 5min --> -0.1C/s to 20C --> hols at 20C, 5min and put on ice
Sample list
No Sample IDs Conc. (ng/ul) #1 6P-2 1.10 #2 6P-3 1.57 #3 7P-2 1.14
- Note: I will reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally.
- I set up one reaction of sample 6P-2 in a full reaction volume for comparison
- KAPA Hyper Prep reaction set up (exactly in the instruction):
- - 50ul of fragmented DNA
- - 7ul of End repair & dA-tailing buffer
- - 3ul of End repair & dA-tailing enzyme mix
- - Total reaction: 60ul
End repair & dA-tailing
Sample IDs Adaptor Reaction volume Tube # 6P-2 TruS half rxn #1 6P-3 TruS half rxn #2 7P-2 TruS half rxn #3 NTC TruS half rxn #4 6P-2 UMI half rxn #5 6P-3 UMI half rxn #6 7P-2 UMI half rxn #7 NTC UMI half rxn #8 7P-2 TruS full rxn #9 TruS = TruSeq adaptor UMI = customized unmethylated Y-adaptor with UMI Tube #1 and #9 will be compared the effect of total volume.