Mar 29, 2017
Cloned Genomic Library using Illumina Adapters
- Philip D. Weyman1
- 1J. Craig Venter Institute
- Protist Research to Optimize Tools in Genetics (PROT-G)
Protocol Citation: Philip D. Weyman 2017. Cloned Genomic Library using Illumina Adapters. protocols.io https://dx.doi.org/10.17504/protocols.io.hfbb3in
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: March 25, 2017
Last Modified: March 10, 2018
Protocol Integer ID: 5315
Abstract
A protocol to create a genomic library (2-5 kb insert size) to screen for genomic regions functioning as centromeres. Uses the NEBNext Illumina library prep kit from NEB to add adapters to fragments and then the fragments can be efficiently assembled into a vector containing homology to the adapter sequences using Gibson Assembly.
Shear DNA with Covaris Sonication
Shear DNA with Covaris Sonication
To prepare 2-5 kb inserts, use the red miniTUBE according to the Covaris instructions. We used 200 µL of 70 ng/µL DNA.
End Prep DNA
End Prep DNA
Use NEBNext Ultra II kit. The user is referred to https://www.neb.com/protocols/2015/09/16/protocol-for-use-with-nebnext-ultra-ii-dna-library-prep-kit-for-illumina-e7645
Fragmented DNA: 50 µL
NEB Ultra II End Prep enzyme mix: 3 µL
NEB Ultra II End Prep reaction buffer: 7 µL
Pipet up and down with P200 10x
Set up PCR machine for following cycle:
Incubate 30 min at 20 C
Incubate 30 min at 65 C
Hold at 4C until ready to proceed.
Ligation of adapters
Ligation of adapters
End Prep Reaction from step 2: 60 ul
Ligation master mix: 30 ul (note, be careful pipeting viscous solution)
ligation enhancer 1 ul
Adapter (undiluted) 2.5 ul
Pipet up and down with P200 10x
Set up PCR machine for following cycle with no heated lid:
20 C for 15 min
Add 3 ul USER enzyme
mix well.
incubate 15 min at 37 C, lid at 50 C.
Cleanup
Cleanup
Add 15 ul Ampure XP beads
Pipet up and down 10x wit P200
Incubate 5 min at Room Temp
Incubate 5 min on Magnet
Wash beads 2x with 200 ul fresh 80% ethanol (keeping tubes on magnetic stand), 30 sec per wash.
Spin briefly on centrifuge and remove remaining ethanol from bottom of tube with pipet
Replace tubes on stand
Air dry on stand with cap open (5 min)
Add 23 ul elution buffer (Qiagen buffer EB = 10 mM Tris, pH 8)
Mix well, incubate 5 min room temp.
Replace on magnet, incubate 5 min.
Remove 20 ul to new tube.
PCR
PCR
Use PCR reagents from kit:
DNA: 15 ul
Q5 master mix 25 ul
Index primer 1: 5 ul
Universal primer 5 ul
Cycle:
98 C, 30 sec
8 cycles of (98 C, 10sec), (65 C 2.5 min)
65 C, 5 min
4 C, hold
Cleanup 2
Cleanup 2
Follow Step 4 cleanup procedure with the following modifications
Add 40 ul Ampure XP beads to 50 ul PCR reaction
Follow the rest of the protocol from step 4 and elute in 20 ul elution buffer.
Design primers for vector
Design primers for vector
After adapter ligation, the DNA fragments should have the following sequences at the ends:
5'-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-
-N2000-5000N-
-GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT-3'
The simplest way to design primers for a vector that will assemble the adapter-ligated fragments into a vector is to use a sequence analysis program (Benchling, etc), paste the above sequence into the vector sequence at the designed place, and design vector primers with ~30-bp overlaps to the above sequence.
After designing primers, amplify the vector, treat with DpnI to remove any vector template, and clean up the reaction with Ampure beads as in step 6.
Gibson assembly
Gibson assembly
The cleaned up library can now be assembled into the cleaned up vector. Try a small amount to start with. Add ~20 fmole each component (vector and insert) in a tube with an equal volume of 2x Gibson Assembly Mix (NEB). Assemble for 1 hr at 50 C. Transform suitable E. coli strain with 1 ul of the assembly. Use highly electrocompetent E. coli (I use Epi300 from Epicentre). Count colonies and calculate how much the assembly/transformation must be scaled up so that you can achieve the number of library colonies required for your application.
Test 10-20 colonies from the preliminary library by colony PCR to make sure they have the proper size insert and that empty vectors are low.