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    Protocol for T7 Library Cloning
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    Protocol for T7 Library Cloning

    Introduction

    Materials

    • DNA Purification Kits
      • Qiagen Lambda Kit (can buy Qiagen Maxi Plasmid Maxi DNA Purification Kit #12162 and make remaining buffers)
      • Gel Purification Kit: (Macherey–Nagel #740609.50) [store at room temperature]
    • PCR Components
      • Herculase II Polymerase: Store at 4°C (GRCF #600677) [store at -20°C]
      • Herculase 5x Buffer: [store at -20°C]
      • dNTP: [store at -20°C]
      • Primers
      • Template DNA
    • Digest
      • 8-well PCR strips
      • Restriction enzymes and buffer
    • Gel
      • Lithium borate
      • Agarose
      • SYBR Safe DNA gel stain (Thermo S33102)
      • 1kb plus DNA ladder
      • Qubit HS Assay Kit (Thermo Q32854)
      • blue light transilluminator (Thermo G6600)
    • ligation
      • T4 DNA Ligase (2M units/mL) (NEB M0202M)
    • qpcr
      • Brilliant III Ultra-Fast SYBR QPCR MM (Agilent 600882)
    • Packaging Kit
      • T7Select Packaging Kit: (EMD Millipore #70014-3)

    Procedure

    • Vector Prep
    1. Follow Qiagen Lambda Handbook protocol to prepare vector DNA.
    1. Quantify eluted DNA via nanodrop.
    • Insert Prep
    1. PCR amplify insert according to the thermocycler protocol below.
    • The number of reactions may be modified based on expected insert yield. Be wary of increasing the number of PCR cycles as it is important to avoid reagent depletion.
    Table1
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    A
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    D
    1
    2
    		Reactions120
    3
    		Volume20400
    4
    		ReagentProtocol
    5
    		Water14.52901) 95 degC; 2 m
    6
    		5x Herculase Buffer4802) 95 degC; 20 s
    7
    		100 mM DNTP0.243) 58 degC; 30 s
    8
    		100 uM Primer_F0.0514) 72 degC; 30 s
    9
    		100 uM Primer_R0.0515) Go to (2) 29x for 30 cycles
    10
    		Herculase0.246) 72 degC; 3 m
    11
    		1 ng/uL TWIST library pool (1 uL Iibrary pool + 99 uL Elution Buffer)1207) 4 degC; hold
    12
    		sum20400
    12 rows
    1. Pool reactions for each insert and cleanup with a Macherey-Nagel kit. Follow the Macherey-Nagel protocol provided in the kit except for the following: to elute, add 50 uL of elution buffer and incubate at 37C for 10 minutes prior to centrifugation. After centrifugation, transfer the eluted volume again to the top of the column membrane, incubate at 37C for an additional 10 minutes, and elute again by centrifugation. Quantify via nanodrop.
    1. If a large number of PCR reactions are pooled, a single cleanup may be insufficient to purify the insert DNA. Therefore, a second cleanup is recommended. Perform the double-elution procedure described in the previous step. Quantify via nanodrop.
    • Digest
    1. Set up as many 50 uL digest reactions for your insert and vector as possible based on the nanodrop concentration. Per each 50 uL reaction, load 1 ug of sample (either twice-column-purified insert or Lambda prep vector DNA), as described in the example below for a yield of 50 uL of 200 ng/uL template. Save a small amount of each undigested template to compare to your digested products via gel electrophoresis.
    Table2
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    A
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    1
    Example Digest
    2
    		template ng/uL200
    3
    		reactions110
    4
    		reaction volume (uL)50500
    5
    		H2O36.00360.00
    6
    		Cutsmart (10x)550
    7
    		Template5.0050.00
    8
    		HindIII220
    9
    		EcoRI220
    10
    		incubate at 37C overnight (16 hours)
    10 rows
    1. After incubating the digest reactions at 37C overnight, pool reactions based on the identity of their DNA template and use a Macherey-Nagel kit to cleanup and concentrate your digested insert and vector. Each column can purify a maximum of 25 ug. If necessary, load your pooled volumes across multiple columns. Perform the double-elution procedure described in a previous step. Quantify via nanodrop.
    • Gel Extract
    1. Pour a 100 mL lithium borate gel with 2% agarose and 10 uL of SybrSafe stain and use a comb to accommodate 60 uL of sample + dye in each lane. After the gel is set, fill the chamber with 1x lithium borate.
    • For the vector, consider using a lower % agarose (<1%) and long electrophoresis run times as an additional purification measure - to separate digested vector from any remaining undigested vector.
    1. Mix each digested and undigested sample with 10x OG loading dye (treat the 10x dye as 5x), and load samples onto the gel, along with a 1 Kb Plus DNA ladder.
    1. Run the gel at a constant 200 V for 30 minutes.
    1. Visualize the gel on a Blue Light Transilluminator (do not use UV light). Take a picture, then extract each digested band using a clean razor each time.
    1. Use a Macherey-Nagel kit to cleanup the gel extract bands. Perform the double-elution procedure described in a previous step. Elute the vector in 30 uL water instead of elution buffer, and elute the insert(s) in 30 uL of elution buffer or less instead of 50 uL. Quantify the vector via nanodrop and quantify the inserts via qubit.
    1. If the gel-extracted vector has a concentration less than 500 ng/uL, use a speed-vac to increase the concentration.
    • Ligate
    1. Setup as many 5 uL ligation reactions as possible using 1 uL of ligase per 500 ng of vector and a 3:1 molar ratio of insert to vector.
    1. Run a no insert control, and for each insert run a no ligase, no vector control as well. As a positive control, load 500 ng of an undigested purified DNA of similar length to your intended ligation product. You may scale up the 3:1 Ligation reaction as needed to produce adequate packaged phage.
    Table4
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    F
    G
    H
    1
    		Example Ligation
    2
    		3:1 LigationNo InsertNo LigaseNo VectorPositive Control
    3
    		Volume55555
    4
    		Water0.952.571.951.882.93
    5
    		10xLigase Buffer0.50.50.50.50.5
    6
    		ng/uL vector5400.930.930.9300
    7
    		ng/uL insert5Digested Insert1.6201.621.620
    8
    		ng/uL positive control DNA870Undigested Positive Control DNA00000.57
    9
    		T4 Ligase11011
    10
    		incubate 16C on thermocycler overnight
    10 rows
    • qPCR
    1. After incubating ligations at 16C overnight, perform qPCR using Sybr Green. Take 1 uL from each ligation and perform serial 10-fold dilutions up to a dilution of 10^6. To create a standard curve, also perform serial dilutions on a stock of your genome control that was also used for the ligation positive control earlier. Load 5 uL of each sample and standard dilution from 10^2 to 10^6 for qPCR analysis.
    Table3
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    A
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    D
    1
    		Reagent1x 10 uL Reaction (uL)50Protocol (Applied Biosystems)
    2
    		2x SYBR mix52501) 95 degC; 10 min
    3
    		100 uM Primer_F0.0212) 95 degC; 20 sec
    4
    		100 uM Primer_R0.0213) 58 degC; 30 sec
    5
    		0.1 mM Dye0.0214) 72 degC; 60 sec
    6
    		sum5.063035) Go to (1) 40x
    7
    		6) Dissociation
    7 rows
    1. Compare CTs to approximate the relative concentrations of your mock ligation, ligations, and no-insert control. Ideally your ligation concentrations are very close to the mock ligation concentration (>90%) and the no-insert control is much less concentrated (<5%). Other negative controls should have very low approximate concentrations.
    • Package
    1. If ligations look good by qPCR, package using T7Select Packaging Kit. Thaw packaging extract on ice.
    • It is important that ligations be packaged as soon as possible after validation by qPCR. Packaging efficiency seems to dramatically decrease (to 25-50% of original) by storing a ligation reaction at 4C for even one additional day. It may be possible to freeze a ligation reaction at -80C overnight and package the next day, but we have not yet thoroughly validated this method.
    1. In 6 uL reactions (5 uL packaging extract + 1 uL ligation product), package your no-insert and positive control conditions for assessment by plaque assay. Add ligation to packaging extract in a ratio 1:5 by volume. Incubate at 22C for 2 hours.
    1. Quench with LB. Per 5 uL of packaging extract, quench with 54 uL of LB. Mix gently and thoroughly.
    • Plaque Assay
    1. Perform a plaque assay as described in the T7Select Handbook. In addition to titering your ligation reactions, titer the no-insert condition to establish a background value, and titer the POU3F1 condition to characterize packaging efficiency. We have observed titers between 5e6 pfu/mL and 1e7 pfu/mL after quenching packaging reactions as described above.
    1. Once a sufficient quantity of pfu has been obtained, expand the library according to the library expansion protocol. Then, expand the phage extracted from this first expansion once more to produce phage that will be used for PhIP-seq screens.

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