Meridian’s Glycerol-Free NGS Library Preparation Solution Overview of library preparation by fragmentation and relevant Meridian products to perform each step Fragments with 5’ and 3’ overhangs are repaired to create blunt ends, these are then phosphorylated and A-tailed, leaving ends amenable to adapter ligation. Sequencing adapters are then ligated to create complete sequencing-ready library fragments.
5’ 3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
FRAGMENTATION
STEP 1
5’
5’
3’
3’
5’ 3’
5’ 3’
Lyophilized NGS Enzymatic DNA Fragmentation Kit (MDX224) SEE PAGE 3
3’
5’
5’
3’
5’
5’
5’
5’
3’
3’
5’ overhang 3’ 3’
3’ overhang
5’ 3’ 5’ 3’ 5’ 3’
5’ 3’ 5’ 3’
3’
3’
5’
5’
5’
5’
3’
3’
5’ overhang
5’ overhang 5’ overhang
3’ overhang 3’ overhang 3’ overhang
5’
5’
3’
3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
3’
5’ 3’ 5’ 3’ 5’ 3’ 5’
5’
3’
5’ 5’ 5’ 3’
5’ 5’ 5’ 3’
3’ 3’
3’ 3’
5’ overhang
3’ overhang
5’ 3’
5’ 3’
5’ 3’
5’ 3’
5’ 3’
CREATE BLUNT ENDS
STEP 2
(3’ – 5’ exonuclease activity)
(Fill in 3’ – 5’ direction)
3’ 3’
5’ 5’
5’ 5’
3’ 3’
5’ 5’ overhang 5’ overhang 5’ overhang P 3’
3’ overhang 3’ overhang 3’ overhang 5’ 3’ 5’ 3’
Glycerol-Free T4 DNA Polymerase (HC) (MDX207) SEE PAGE 4 Glycerol-Free DNA Pol I Klenow Fragment (HC) (MDX208) SEE PAGE 4
5’
3’ 5’ 3’
(3’ – 5’ exonuclease activity) (3’ – 5’ exonuclease activity)
(Fill in 3’ – 5’ direction) (Fill in 3’ – 5’ direction)
5’ 3’
5’ 3’
5’
5’
3’
3’
5’ 3’ 5’ 3’
5’ 3’ 5’ 3’
5’ 3’ 5’ 3’
(3’ – 5’ exonuclease activity) (3’ — 5’ exonuclease activity)
(Fill in 3’ – 5’ direction) (Fill in 3’ — 5’ direction)
P
P
5’
5’
3’
5’
P
3’
3’
(3’ – 5’ exonuclease activity) (3’ – 5’ exonuclease activity) (3’ – 5’ exonuclease activity) 5’ 3’ 3’ 5’ P 5’ P
P 5’ (Fill in 3’ – 5’ direction) (Fill in 3’ – 5’ direction) (Fill in 3’ – 5’ direction) 5’ 3’
5’ 3’
5’ 3’
3’
3’
P
5’
PHOSPHORYLATION
STEP 3
A
Glycerol-Free T4 Polynucleotide Kinase (HC) (MDX206) SEE PAGE 5
3’
P P
5’ 5’
3’
3’
5’
P 3’
P 5’ 5’
3’ 3’
A
P A
A
5’
3’
3’ 3’ P 5’ P 5’ P 5’ A- 5’ A 3’ 3’ A 3’ -T A -T
P 3’
-A 5’ 5’ 5’ 5’ 5’ A A
P P 3’ P
3’ 5’
P
A
-T
5’
3’ 3’ P
A-TAILING
STEP 4
A-
T- A 5’
P
Glycerol-Free Taq HS High Conc. DNA Polymerase (MDX170) SEE PAGE 6
Adaptor -A T- -A T-
3’ 3’
Adaptor
A A P 5’ A- 3’ A -T A-
P P
3’
Adaptor
Adaptor
Adaptor
Adaptor
-A
T-
5’
P
Adaptor
-T -T A-
Adaptor -A
Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adapter
Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adaptor Adapter
ADAPTER LIGATION
STEP 5
A- A-
T- T-
-A
Glycerol-Free T4 DNA Ligase (HC) (MDX200) SEE PAGE 6
-T
-A
T-
Glycerol-Free High-Fidelity Pfu (HC) (MDX203) SEE PAGE 7 STEP 6 LIBRARY AMPLIFICATION
2
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