DeLong:Lab Protocols:MidSizeInsert Plasmid Library

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

-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

[1] prepare pMCL200 DNA

digest with EcoRV to get blunt-end linear vector PCR cleanup to remove enzyme treat with shrimp alkaline phosphatase gel purify.

[2] shear environmental DNA

-using GeneMachines Hydroshear (Genomic Solutions) -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

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) [should calibrate shearing orifice per application] 25 cycles chill on ice immediately after shearing

[3] end-repair insert DNA

-use repair-it from Epicentre (includes T4 DNA polymerase and T4 polynucleotide kinase) to get blunt, 5’ phosphorylated ends

8 µL 10X End-Repair Buffer 8 µL 2.5 mM dNTP mix 8 µL 10 mM ATP 4 µL End-Repair enzyme mix 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 (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

[4] gel purify insert DNA

run sample on 1% LMP SeaPlaque agarose-TAE gel in 1X TAE chill gel at 4º C before use load entire sample in one lane run ~100 V (for large gel) 3-4 hrs; change running buffer after 2 hrs use 1 µL (500 ng) 1 kb DNA ladder as marker

stain with SYBRGold 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

gel purify with QIAEX II kit (see manual, no more than 150 mg gel/tube, don’t vortex if any fragments >10 kb, elute in buffer EB [10 mM Tris-Cl, pH 8.5])

or

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

[5] 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 0.5 µL T4 CNA ligase (400,000 units/mL so ~200 units ligase) ~10 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

[6] transformation and test plates

- Invitrogen Top10-F’ cells work well here (lacIq allele on F’ plasmid with TetR), or use lacIq E. coli strain of your choice

electroporate 1 µL ligation product (10%) into 50 µL electrocompetent cells 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

[7] 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 0.25 µL BamHI 0.25 µL HindIII

~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

[8] plate library for automated picking

-library picking done with Genetix QPixII robot (Genetix, Hampshire, UK)

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 and plate with large beads) 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)