General outline of BAC cloning protocol:
- Order 3 BACs containing the gene of interest. Design primers for screening for presence of BACs, overlap PCRs, screening for integration of shuttle vector into BACs, and sequencing.
- When BACs arrive, screen to confirm presence of your gene. Keep a frozen stock of the BACs.
- Make the BACs electrocompetent and transform with pDelsac to delete SacB. Screen colonies to pick the ones that have deleted SacB. While you’re doing this you can start working on constructing the shuttle vectors.
- Sequence shuttle vector.
- Make the BACs that have totally resolved the pDelsac vector electrocompetent again and transform with the shuttle vector. Screen colonies to pick the ones that have integrated the shuttle vector.
- Purify modified BACs from large scale liquid cultures. Confirm modification by Southern blot.
Supplies:
o BAC clones – order from CHORI.
o Primers – from IDT.
o Plasmid containing insert (for DTR-GFP – p1308).
o pDelsac23/pDelsac24.
o PIR2 One Shot – Invitrogen.
o Supplies for bacterial cultures
o Supplies for LB and LB plates, SOC medium
o Supplies for M9 minimal media plates – see recipe
o Chloramphenicol
o Ampicillin
o PCR supplies
o Enzymes – Taq and Pfu Turbo – Cell culture facility.
o 10 mM dNTPs
o Supplies for making electrocompetent cells
o Sterile 10% glycerol (1 L distilled water, 100 ml sterile 100% glycerol can be purchased from Cell culture facility)
o Qiagen kits: Gel extraction, miniprep, midi/maxiprep
o Supplies for purifying modified BACs – see protocol
o Restriction enzymes: NotI, AscI, any enzymes used for Southern blots – NEB
After receiving BAC clones, miniprep BAC DNA and screen to confirm the gene of interest is present.
· Streak bacteria from LB slab on LB + chloramphenicol plate, incubate at 37۫ overnight.
· Pick a single colony from each plate and transfer to 3 ml LB + chloramphenicol liquid culture. Shake overnight at 37.
· Miniprep BACs using modified protocol from CHORI website:
o Transfer 1 ml from culture to eppendorf tube, spin 5 min at max speed.
o Remove supernatant, transfer an additional 1 ml to eppendorf tube and spin again.
o Remove supernatant. Resuspend pellet in 0.3 ml P1 (50 mM Tris, pH 8; 10 mM EDTA; 100 ug/ml RNase A – filter sterilized) by vortexing.
o Add 0.3 P2 (0.2 NaOH; 1% SDS – filter sterilized). Mix by inverting tube. Let sit at room temperature 5 min. Suspension should become translucent.
o Slowly add 0.3 ml P3 (3M KOAc – autoclaved) and mix by inverting. Place on ice at least 5 min. A white precipitate will form.
o Spin at 10,000 rpm, 4 C for 10 min.
o Place tubes on ice. Transfer supernatant to a tube containing 0.8 ml ice-cold isopropanol, avoiding the precipitate. Mix by inverting. Place on ice at least 5 min.
o Spin at 10,000 rpm, 4 C for 15 min.
o Remove supernatant. Wash with 0.5 ml 70% ethanol. Spin at 10,000 rpm, 4 C for 5 min.
o Remove supernatant. Air dry pellet, resuspend in 40 ul TE. Don’t pipet up and down to resuspend – allow solution to sit and tap the tube.
· Freeze the leftover 1 ml of bacteria at -80 C. (Spin down, remove supernatant, and resuspend in 0.5-1 ml LB + 10% glycerol + chloramphenicol).
· PCR screen:
o 19 ul dH20
2.5 ul 10x PCR buffer
0.75 ul 50 mM MgCl2
0.5 ul 10 mM dNTPs
0.5 ul F primer
0.5 ul R primer
0.25 ul Taq
1 ul template
o Primers: One reaction with FasL F1/R1, one reaction with FasL F2/R2.
o PCR program:
95 – 2 min
(95 – 30 sec, 60 – 30 sec, 72 – 1 min) x 30
72 – 7 min
4 – hold
Shuttle vector construction:
o PCR to produce Box A, Box B, and DTR-GFP insert:
o Box A/Box B:
§ 40 ul dH20
5 ul 10x Pfu buffer
1 ul 10 mM dNTPs
1 ul Box A or B F primer
1 ul Box A or B R primer
1 ul Pfu turbo
1 ul template (BAC miniprep DNA)
§ PCR program:
95 – 2 min
(95 – 30 sec, 60 – 30 sec, 72 – 1 min) x 30
72 – 7 min
4 – hold
o DTR-GFP insert:
§ 40 ul dH20
5 ul 10x Pfu buffer
1 ul 10 mM dNTPs
1 ul FasL-DTR F primer
1 ul FasL-DTR R primer
1 ul Pfu turbo
1 ul template (p1308 plasmid)
§ PCR program:
95 – 2 min
(95 – 30 sec, 60 – 30 sec, 72 – 2 min) x 30
72 – 7 min
4 – hold
o Gel purify PCR products (Qiagen kit). Use long wave UV lamp when cutting bands. Elute in 30 ul EB or water.
o Run gel with 5 ul of each PCR product to check the relative concentrations of each product.
o Overlap PCR:
o Template: equal amounts of Box A, Box B, and DTR-GFP insert (ie, if Box A band is twice as bright as Box B, the volume of Box A used as template should be half the volume of Box B used as template). Total volume of templates should be 15-20 ul per reaction.
o 15-20 ul templates
1 ul Box A F primer
1 ul Box B R primer
5 ul 10x Pfu buffer
1 ul 10 mM dNTPs
1 ul Pfu turbo
dH20 to total volume of 50 ul/reaction
o PCR program:
95 – 2 min
(95 – 30 sec, 60 – 30 sec, 72 – 3 min) x 30
72 – 7 min
4 – hold
o Gel purify overlap PCR products. Elute in 30 ul EB or water.
o Digest entire volume of gel purified overlap PCR product with AscI and NotI (100 ul total volume, NEB buffer 4 + BSA, 5 ul each Not I and AscI; digest overnight).
o Digest pDelsac23 with NotI and AscI (100 ul total volume, NEB buffer 4 + BSA, 5 ul each Not I and AscI, 10 ul = 1 ug plasmid; digest overnight).
o Gel purify digests. Elute in 30 ul EB or water.
o Run gel with purified digests to check relative concentrations.
o Ligate vector and insert overnight.
o 20 ul total volume: equal amounts of vector and insert with combined volume of up to 16 ul, 2 ul 10x ligation buffer, 2 ul ligase. Incubate in 16 C water bath overnight.
o Transform Pir2 cells with shuttle vector.
o Thaw tube of Pir2 cells on ice.
o Pipet 1-5 ul ligation reaction into tube. Tap gently.
o Incubate 30 min on ice.
o Heat shock – 30 sec in 42 C water bath, then return to ice.
o Add 250 ul prewarmed SOC. Shake tube at 37 C for 1 hr.
o Spread bacteria on LB + ampicillin plates.
§ Use several plates with different volumes of bacteria (ie 20 ul, 50 ul, 200 ul).
o Pick several single colonies and transfer to 3 ml LB + ampicillin liquid culture. Shake at 37 C overnight.
o PCR to confirm presence of insert
o 38 ul dH20
5 ul 10x PCR buffer
1.5 ul MgCl2
1 ul 10 mM dNTPs
1 ul DTR F1 primer
1 ul DTR R1 primer
0.5 ul Taq
2 ul from bacterial culture
o PCR program:
95 – 2 min
(95 – 30 sec, 60 – 30 sec, 72 – 1 min 30 sec) x 30
72 – 7 min
4 – hold
o Transfer cultures with positive PCR results to 100 ml LB + ampicillin liquid culture. Shake at 37 C overnight.
o Midiprep shuttle vectors (Qiagen kit).
o Send shuttle vectors to sequencing.
Making BAC clones electrocompetent:
o Streak 5 ul frozen BAC clone on LB + chloramphenicol plate. Incubate at 37 C overnight.
o Pick a single colony and transfer to 3 ml LB + chloramphenicol liquid culture. Shake at 37 C overnight.
o Transfer 1 ml to 100 ml LB + chloramphenicol liquid culture. Shake at 37 C until OD600=0.4. (Usually 2-3 hours.)
o Transfer to 50 ml conical tubes (2 tubes per 100 ml culture, only fill to 45 ml). Place on ice 10 min.
o Spin 15 min at 6000 rpm, 4 C in ____ rotor. (The tubes don’t fit well in the rotor. Remove lids from tubes and press them into the rotor. To remove the tubes, put the lids back on and use forceps as a lever to pry them out.)
o The rest of the protocol must be done in the 4 C cold room:
o Carefully remove supernatant without dislodging pellet. Resuspend in 25 ml 10% glycerol. Spin 15 min at 6000 rpm, 4 C.
o Repeat glycerol resuspension/spin two more times. (Pellet is very prone to being dislodged at this stage.)
o After final spin, remove as much supernatant as possible and transfer pellet + any left over supernatant to an eppendorf tube. Spin in benchtop centrifuge at maximum speed 10 min (4 C). Remove all supernatant and resuspend pellet in 200 ul 10% glycerol.
§ (One 100 ml culture = two 50 ml conical tubes = 2 x 200 ul in glycerol).
o Store electrocompetent cells at -80 C.
Transforming electrocompetent cells (with pDelsac or shuttle vector):
o Thaw electrocompetent BAC cells on ice. Prepare an eppendorf tube containing 1ml room temperature SOC. Place cuvette on ice.
o Pipet 1 ul plasmid onto upper part of inside wall of cuvette (not inside the well). Pipet 50 ul electrocompetent cells on top of the 1 ul plasmid. Replace cuvette lid and tap side of cuvette to get cells and plasmid into the well.
o Make sure cuvette is dry. Place in electroporator and tighten.
o Electroporate: 1800 V, 250 W, 50 uF. Press pulse.
o Immediately pipet 500 ul from SOC tube into cuvette and resuspend cells.
o Transfer contents of cuvette to SOC tube, then transfer contents of SOC tube to capped tube. Shake at 37 C for one hour.
o Spread contents on tube on LB + chloramphenicol + ampicillin plates (3 plates with 200 ul each, 1 plate with 50 ul). Incubate at 37 C overnight.
o ***If you see sparks, the lid pops off the cuvette, or the time constant is not approximately 11.5-12 ms, the ionic strength of the electrocompetent cells or the plasmid is too high. Try washing the cells 1-2 more times with 10% glycerol, or cleaning up the plasmid with a gel extraction/PCR purification column (elute in dH20 instead of EB).
Screening for incorporation of pDelsac:
o Pick several single colonies from LB + chloramphenicol + ampicillin plates and resuspend each colony in 30 ul LB + chloramphenicol + ampicillin liquid culture.
o PCR screen the liquid cultures – two reactions for each one (Reaction 1 primers = RP1 and RP3, Reaction 2 primers = RP2 and RP4-23 or RP4-24 depending on whether pDelsac23 or pDelsac24 was used).
38 ul dH20
5 ul 10x PCR buffer
1.5 ul MgCl2
1 ul 10 mM dNTPs
1 ul primer 1
1 ul primer 2
0.5 ul Taq
2 ul from 30 ul liquid culture
o PCR program:
95 – 2 min
(95 – 30 sec, 60 – 30 sec, 72 – 1 min 30 sec)
72 – 7 min
4 – hold
o Wild type: RP1/RP3 = 1.6 kb, RP2/RP4-23 = 1.2 kb
o Recombined: RP1/RP3 = 1 kb, RP2/RP4-23 = 730 bp
o Pick cultures that have resolved one box (one reaction has WT band only and the other reaction has recombined band only). Spread on M9 minimal media plates. Incubate at 37 C for 3-4 days. Repeat PCR screen and select colonies that have resolved both boxes (both reactions have recombined band only). Freeze a stock of these colonies at -80 C and make them electrocompetent to transform with shuttle vector.
Screening for integration of shuttle vector:
o After transformation with shuttle vector, repeat the above procedure using the following primer pairs: Reaction 1 = Box A US F1 + DTR R3, Reaction 2 = DTR F3 + Box B DS R1.
Purification of modified BACs from large culture:
o Shake 500 ml culture at 37 C overnight.
o Centrifuge bacteria 15 min at 5000 rpm 4 C.
o Remove supernatant and resuspend in 20 ml 10 mM EDTA pH 8.0. Incubate 5 min at room temperature.
o Lyse with 40 ml 0.2 N NaOH + 1% SDS for 5 min at room temperature.
o Neutralize with 30 ml cold 2 M KOAc (50 ml 7.5 M KOAc, 23 ml glacial acetic acid, 127 ml dH2O) for 5 min on ice.
o Spin 15 min, 10000 rpm, 4 C.
o Mix supernatant with equal volume isopropanol.
o Spin 15 min, 5000 rpm, 4 C.
o Dissolve pellet in 9 ml 10:50 TE, then mix with 4.5 ml 7.5 M KOAc.
o Incubate at -80 C for 30 min, then thaw.
o Once thawed, spin 15 min, 6000 rpm, 4 C.
o Precipitate supernatant with 2.5 volumes ethanol.
o Spin 15 min, 8000 rpm, 4 C.
o Resuspend pellet in 700 ul 50 mM Tris-Cl pH 7.6 + 50 mM EDTA pH 8.0.
o Add 150 ug/ml RNase A.
o Incubate 30 min at 37 C.
o Add equal volume phenol:chloroform. Shake tube, then spin in benchtop centrifuge at max speed 5 min.
o Transfer aqueous phase to a new tube. Add equal volume choloroform. Shake tube, spin again.
o Transfer aqueous phase to new tube. Precipitate by adding equal volume of isopropanol.
o Wash with 70% ethanol.
o Air dry pellet. Resuspend in 100 ul TE, and store at 4 C.
M9 minimal media plate recipe:
o Autoclave 131.5 ml dH20 containing 3 g agar and a stirbar for 30 min.
o While autoclaving, prepare 50 ml 5x M9 salts:
o 1.7 g Na2HPO4
0.75 g KH2PO4
0.125 g NaCl
0.25 g NH4Cl
o Add 50 mL dH20, then filter sterilize.
o Place M9 salts, 50% sucrose (sterile), and 10% glycerol (sterile) in 55 C water bath.