DeLong:Lab Protocols:MidSizeInsert Plasmid Library

Construction of Mid-Size Insert Environmental Genomic Expression Libraries in Plasmid pMCL200

Adapted from Joint Genome Institute protocols for library construction and Epicentre protocols for CopyControl Fosmid library construction (Epicentre, Madison, WI).

contributed by Jennifer Braff

• pMCL200 has p15A ori; 20-40 copies/cell, 2535 bp, CamR.
• MCS under control of wild type lac promoter.
• vector has blue/white screen for presence of insert.

shear environmental DNA

• if using crude extracts of environmental DNA, filter DNA through a 0.2 µm filter then concentrate before shearing.
• wash Hydroshear before and after use as directed with filter sterile 4X HCl, 4X NaOH, and 4X TE.
• for precise fragment size, each shearing orifice should be calibrated before use.

dilute DNA to ~100 ng/µl in filter sterilized TE

incubate 30 minutes at 37º C; vortex every 10 minutes

spin 20 minutes at 14 K (any pellet indicates incomplete resuspension)

transfer sample to a clean tube

run 5-10 µg DNA (if available) in 50–150 uL sample

shear at speed code 15 (gives 7-10 kb chunks) or SC 16 (gives 8-12 kb chunks) on GeneMachines Hydroshear (Genomic Solutions)

25 cycles

chill on ice immediately after shearing

end-repair insert DNA

8 µL 10X End-Repair Buffer

8 µL 2.5 mM dNTP mix

8 µL 10 mM ATP

4 µL End-Repair enzyme mix (End-It DNA End-Repair kit, Epicentre)

52 µL sheared DNA

[total volume = 80 µL; scale up as needed)

1 hour at room temperature (don’t leave longer as DNAP can chew ends)

add 7 µL 125 mM EDTA (to 10 mM) to prevent DNAP from chewing ends during heat inactivation

70º C for 10 minutes to heat inactivate

chill on ice

T4 polynucleotide kinase may not be completely heat inactivated, though the enzyme should be removed by gel purification. If kinase carry-over and phosphorylation of vector DNA during ligation is a concern, the reaction can be phenol-chloroform extracted.

EtOH precipitation of DNA to concentrate:

add 2 µL Pellet Paint (Novagen) (be sure to resuspend well)

add 1/10 volume 3 M NaAcetate (pH 5.2) to sample and mix

add 2 volumes of 95% EtOH, invert to mix

incubate 2 minutes at room temperature

spin 14K XG, 5 min

remove sup’n with pipette

wash with 70% EtOH, spin and remove sup’n as above

wash with 95% EtOH, spin and remove sup’n as above (careful of pellet here)

dry 10 minutes and resuspend in 30 uL TE

gel purify insert DNA

run sample on 1% LMP SeaPlaque (FMC BioProducts ) agarose-TAE gel in 1X TAE

chill gel at 4º C before use

load entire sample in one lane

load 500 ng 1 kb DNA (New England Biolabs) ladder as marker

run ~100 V (for large gel) 3-4 hrs; change running buffer after 2 hrs

stain with SYBRGold (Molecular Probes) 15 minutes with gentle shaking

view on non-UV safe light table and cut smaller (~6-8 kb) and larger (~8-10 kb) fractions of sheared DNA

cut away any excess agarose

if fragements 10 kb or larger, gel purify with QIAEX II kit (Qiagen) (no more than 150 mg gel/tube, don’t vortex samples)

or

if fragments <10 kb, gel purify with QIAQuick kit (Qiagen) (do isopropanol and QG washes, let PE sit 5 minutes and repeat PE wash, elute with 50 µL warm [50º C] EB buffer, let sit 10 minutes before eluting)

run a gel to check vector and insert concentrations and allow calculation of a more accurate insert:vector molar ratio in the ligation step (don’t skip this step)

blunt-end ligation

• shoot for 10-15 ng of vector DNA in a 10-15 µL ligation reaction (can up this if sufficient insert is available).
• an insert:vector molar ration of approximately 3:1 works well here.
• don’t use quick ligase.

1 µL 10X T4 DNA ligase buffer

200 units T4 DNA ligase

~15 ng blunt-end EcoRV cut, phosphatase-treated, gel-purified pMCL200

insert DNA prepared as above

to 10 µL with stH20

16 C o/n

heat inactivate 65 C for 10 minutes

dialyze against water for 30 minutes on Millipore 0.25 µm filter

transformation and test plates

• Top10-F’ (Invitrogen) cells used below carry a lacIq allele and TetR marker on an F’ plasmid to permit IPTG inducible gene expression, or use a lacIq E. coli cloning strain of your choice.

electroporate 1 µL ligation product (10%) into 50 µL Top10-F' electrocompetent cells (Invitrogen)

25 µF, 2.0 kV, 200 Ohms, 1 mm cuvette

mix w/ 1 mL room temp SOC

37º C with shaking for 1 hour

plate 10 µL transformation product diluted 1:20 into SOC (200 µL total volume) onto a small LB-CAM/Tet/X-gal plate and the same onto a LB-CAM/Tet/X-gal/IPTG plate (see below for reagent concentrations)

store the remaining transformation product as a 25% glycerol stock at -80º C to plate once library yield and quality are confirmed

incubate test plates o/n at 37º C

do colony count to estimate yield

score % blue (likely no-insert) colonies

quality check

pick 6-10 white colonies from the LB-CAM/Tet/X-gal/IPTG test plate

grow up o/n liquid cultures and prep plasmid DNA (4 mL of culture is plenty)

digest prepped DNA to check insert presence/length

2 µL NEB buffer 2

5 units BamHI (New England Biolabs)

5 units HindIII (New England Biolabs)

200 ng plasmid DNA

H20 to 20 µL

37º C for 4 hours

65º C for 20 minutes

run out on 1% agarose gel w/ EtBr to visualize bands

plate library for automated picking

• library picking done with QPixII robot (Genetix).

pour large rectangular LB-CAM/Tet/X-gal/IPTG plates (no bubbles)

plate glycerol stock of transformation <3000 colonies/plate (bring volume to 600 µL with SOC media for plating)

37º C overnight then store 2 days at 4º C to let blue color develop on no-insert clones

pick as for fosmid libraries, except set QPixII to pick only white colonies

working concentrations and stock solutions

chloramphenicol (CAM) used at 12.5 µg/mL; stock is 12.5 mg/mL in EtOH

Tetracycline (Tet) used at 10 µg/mL; stock is 10 mg/mL in 50% EtOH

X-gal used at 40 µg/mL; stock is 40 mg/mL in dimethylformamide

IPTG used at 1 mM; stock is 500 mM in H20 (0.71 g IPTG in 5 mL H20, filter 0.2 µm)