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)
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)
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)
- 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
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)