Revised 15.03.01 Note: Modified this lab protocol to include exactly what we do vew
Mass Excision and Transfection of Unamplified EST Libraries
Ordering library from Stratagene:
Ensure that Stratagene is instructed that we want fraction 1 unamplified, otherwise they will automatically amplify the library using this fraction [one must write a letter and confirm by telephone that this instruction will be followed]. Best is to talk with them when library has been made, but before amplified to see how to proceed.
Receipt of Library:
Immediately make copies of the original documentation arriving with the library. Give one copy to Pat to put in her "custom cDNA libraries" folder. Put the original documentation in the laboratory binder set aside for this purpose. Make as many working copies as you like, but except for making copies never remove original documentation from the laboratory binder.
Confirm that titers provided in the original documentation match those recorded on the samples received from Stratagene. Report any discrepancy to the boss.
Add DMSO to 7% to the amplified library. Store 50 aliquots of 50μl in 0.5ml screw-cap micro-centrifuge tubes (with O-ring), at –80°C. Store the remainder in 1.5ml screw-cap micro-centrifuge tubes (with O-ring), at –80°C in aliquots of 1.0ml. [Note that addition of the DMSO will modestly decrease the titer because of the increase in volume.]
Unamplified material can be kept in darkness at 4°C for up to three months maximum, after which time add DMSO to 7% and store at –80°C. Note that during these three months at 4°C, the titer will slowly decrease. Do not use either the cold room or the reach-in cold cabinets for storage as they are not sufficiently reliable. Use the refrigerator compartment of our household-style refrigerator/freezer. Shield the phage from fluorescent light as much as is practical.
The SOLR and XL1-Blue MRF′ cells and the ExAssist helper phage should be stored at –80°C.
The ExAssist should be stored in aliquots and per Stratagene titer’d with the first thaw, to determine (pfu/ml) than add to 7% DMSO and aliquot. Our protocol is to thaw once in order to aliquot in micro-centrifuge tubes and re-freeze at –80°C. Titer has not decreased appreciably in the past with one thaw. [Note per Stratagene: ExAssist is sent to us in 7% DMSO in which case we would no need to add 7% unless we titer and amplify the phage. Also, Chloroform is not present in ExAssist. It does not kill lambda phage but Chloroform does kill M13 and other helper phages. Per Alicia Pierini Stratagene Technical Services.]
Bacterial stocks: SOLR and XL1-Blue MRF′ cell stocks are thawed once and grown overnight @ 37°C, 250 RPM in fortified LB Broth with respective antibiotics. Dilute the concentrated cell lines to 0.2-0.4 @ OD600 in fortified broth and add respective antibiotics. Continue to grow @ 37°C, 250 RPM to OD600 1.0. Prepare stocks in sterile micro-centrifuge tubes by mixing well 250μl of cells with 250μl of 50% glycerol in water. Freeze and store these tubes at –80°C. Use a tube only once for inoculation, then discard it in the biohazard waste.
- Before proceeding, read thoroughly the information manual provided by Stratagene with the library.
- Test your skill at mass excision. Dilute the amplified library with SM buffer so that its concentration (pfu/ml) is identical to that of the fraction 1 unamplified library fraction. Only after satisfactorily completing this test should you proceed to work with unamplified library phage. This test should be done immediately before excising unamplified material. The protocol to be followed is described on the following pages.
- As soon as possible after receipt, excise and pick the library, testing as quickly as possible 384 randomly picked clones for quality. Upon completion (20,000 picks), add DMSO to 7% to the unamplified library fractions. Store these fractions individually in screw-cap (with O-ring) micro-centrifuge tubes at –80°C.
Mass excision protocol for unamplified libraries:
Before proceeding, ensure that you have successfully completed the test excision described immediately above.
- As much as is possible, always use the same stocks of all reagents/buffers/antibiotics, cells/helper phage, etc., etc. If for any reason you must change or replenish a stock solution, describe exactly what you have done in your notebook. This information is invaluable for learning what has gone wrong if there should be a failure.
DAY 1
Prepare 250ml fortified broth by mixing 220ml LB with 5ml of 10% maltose (final concentration 0.2% maltose) and 25ml of 100mM MgSO4 (final concentration 10mM MgSO4) store at 4°C and use what remains on Day 2. Grow 25ml cultures of XL1-Blue MRF′ and SOLR cells 16 hr overnight @ 37°C, 250 RPM in 25ml of fortified LB broth. To MRF′ cells add 75μl of 5mg/ml Tetracycline (final concentration 15ug/ml Tetracycline) and to SOLR cells add 125ul of 10mg/ml Kanamycin (final concentration of 50ug/ml.)
- Please note that there are special concerns and restrictions on the use of antibiotics for selection, especially tetracycline, which loses its potency very quickly after addition to medium. If you are unaware of these concerns, please obtain this information before beginning.
- Inoculate cultures directly from bacterial stocks in the –80°C freezer, either from ice splinters or thaw and inoculate with a 1ul loop.
- Plan ahead for Day 2 by obtaining titers for the library phage, ExAssist phage and of XL1-Blue MRF′ cells. Calculate as described below in step 3 of Day 2 the excision mix(es) you will need to make.
DAY 2
- Using the HP Diode Array Spectrophotometer, blank Spec with fortified broth w/o antibiotics. Measure and record the OD600 of both cell lines after 16hr growth at 37°C, 250 RPM. The OD600 of the cells will very likely exceed the maximum value of the spectrophotometer.
- Prepare 3 disposable cuvettes, each containing 3ml fortified broth w/o antibiotics as described on Day 1. To 1st cuvette, add 30μl of cells, to 2nd 100μl, and to the 3rd 300μl. Measure and record the OD600 values. From these data calculate what volume of cells you will need to add to 50ml of fortified broth in order to have an OD600 between 0.2 and 0.4, add respective antibiotics. To MRF′ cells add 150μl of 5mg/ml Tetracycline (final concentration 15ug/ml Tetracycline) and to SOLR cells add 250ul of 10mg/ml Kanamycin (final concentration of 50ug/ml.)
- Continue incubating @ 37°C, 250 RPM. Measure the OD600 periodically until it reaches 1.0. Referring to respective cell growth curve can reduce the number of measurements necessary to get to OD600 1.0.
- Transfer 5ml of cells into a sterile Oak Ridge centrifuge tube and collect the cells by centrifugation for 5 min at 1000g. Decant and discard the supernatant into biohazard waste. Add 5ml of 10mM MgSO4 to each tube and gently but thoroughly resuspend the cells.
- Briefly centrifuge the library phage before use in order to separate the from the phage solution. The phage will be in the upper aqueous phase.
- Prepare a mixture of XL1-Blue MRF′ cells, phage library, and ExAssist (do not use VCSM13) helper phage in the ratio of 104 ExAssist pfu to 103 XL1-Blue MRF′ to 1 library phage. Begin by calculating the amounts of each to be added (this step should preferably be done before starting the work on DAY 2 and calculations should be double-checked with someone else in the lab).
- Prepare the mixture in a 2.0ml screw-cap micro-centrifuge tube. Work with 100,000 phage library pfu, except with very, very, very rare exceptions (to be approved in advance by the boss).
As an example, for an unamplified library with a titer of 1.2 × 106/ml, and helper phage with a titer of 2 × 1011 pfu/ml, the mix would be:
[Add the 3 components in the order MRF′, library phage, then ExAssist]
[mix the MRF′ cells before measuring them]
library phage (1.2 × 103 pfu/μl) 83μl (yields 105 pfu)
XL1-Blue MRF′ (8 × 105/μl) 125μl (yields 108 cells)
ExAssist helper phage (2 × 108 pfu/μl) 5.0μl (yields 109 pfu)
Please note that for the library and helper phage, the above volumes are for example only. You must calculate the amount actually needed based upon the titers of the two phage preparations with which you are working.
If you are working with amplified library phage, use 107 instead of 105 as above – but keep the number of MRF′ cells and ExAssist helper phage at 108 and 109, respectively.
- Incubate the mixture for 15 min in a water bath at 37°C to permit the bacteria to adsorb the phage.
- Add 1ml of room temperature LB broth. Do not add antibiotic! Incubate for 2.5 hr (never more than 3 hr!) at 37°C in the shaker-incubator at 250 rpm.
- Incubate in a water bath at 68°C for 20 min.
- Centrifuge 10 min at 1000g.
- Transfer the supernatant into a clean, sterile, 2.0ml screw-cap micro-centrifuge tubes (with O-ring),using a sterile blue pipet tip. Save this tube it now contains the excised phagemids. Store at 4°C for up to 3 weeks and reuse in the following steps as desired. Longer storage at 4°C is OK, but the titer will decline over a period of a very few months.
[If desired, this supernatant can be stored at –80°C for an extended period, but this should be done only if absolutely necessary. ? May need to add DMSO to 7%..ask Lee]
Transfection of SOLR cells:
Transfect SOLR cells with the excised phagemids. For the first attempt, if at all possible, use phagemids that have previously been shown to transfect well as a positive control, and proceed as described below with 2μl of newly prepared phagemids to 200ul SOLR cells to determine excision success.
In a sterile micro-centrifuge screw-cap tube, gently mix 2μl of phagemids (from step 10) with 200μl of SOLR cells prepared as described above through step 4 of DAY 2. [A higher ratio of phagemids to SOLR cells (e.g., 50μl phagemids + 100μl SOLR cells) can be used if the phagemid titer is very low.]
Incubate the mixture in a water bath at 37°C for 15 min.
- If this first attempt is fully successful, inoculate a new SOLR culture from cells prepared earlier in the day in MgSO4. Prepare culture as described in Step 1, Day1 and follow with dilution/incubation protocol beginning at Step 1 Day 2 the following day. Transfection for plating requires a larger quantity of phagemids, however, the ratio of phagemids to SOLR cells remains 2μl to 200μl unless the phagemid titer is low.
Note that it is absolutely essential that transfected SOLR cells be completely plated within 2 days (3 days if absolutely necessary). The SOLR cells die quickly in liquid medium but are viable for many days, even a few weeks, on agar.
Titering the transfected cells:
Mix 20μl of transfected cells from step 2 Transfection of SOLR cells with 180μl of LB with ampicillin (1.5μl of a 100mg/ml stock per ml of LB). Pipette 100μl of the resultant mixture on rectangular plates [50ml of NZY agar containing ampicillin (750μl of a 100mg/ml stock solution per 500ml of agar medium, to yield 150μg/ml final concentration)], together with 70μl of 2% X-Gal (20 mg/ml) and 35μl of 0.1M IPTG. See below for spreading details. Consider this plate to be 0.1×.
Take 63μl of cells from step 1 and mix with 137μl of LB-Amp. Pipette 100μl of the resultant mixture on rectangular plates [50ml of NZY agar containing ampicillin (750μl of a 100mg/ml stock solution per 500ml of agar medium, to yield 150μg/ml final concentration)], together with 70μl of 2% X-Gal (20 mg/ml) and 35μl of 0.1M IPTG. See below for spreading details. Consider this plate to be 0.03Χ.
Take 63μl of cells from step 2 and mix with another 137μl of LB-Amp. Pipette 100μl of the resultant mixture on rectangular plates [50ml of NZY agar containing ampicillin (750μl of a 100mg/ml stock solution per 500ml of agar medium, to yield 150μg/ml final concentration)], together with 70μl of 2% X-Gal (20 mg/ml) and 35μl of 0.1M IPTG. See below for spreading details. Consider this plate to be 0.01Χ.
Repeat this process to produce dilutions of 0.003× and 0.001×.
Incubate agar plates overnight at 33°C.
Count the number of colonies/plate and record your results:
0.1× _____________
0.03× _____________
0.01× _____________
0.003× _____________
0.001× _____________
[If the titer is either too high or too low, the above dilutions can be changed accordingly.]
Optional: If there are sufficient colonies suitable for picking from these test plates, please feel free to do so. If you can obtain 384 colonies in this way, you would then have enough to evaluate the quality of the library.
Production plating of the transfected cells:
Note that for production plating, the volumes in step 2 Transfection of SOLR cells would have been increased in proportion.
Following transfection (steps 2&3 Transfection of SOLR cells), dilute the cells in LB-Amp appropriately to obtain a concentration that will yield 300 to 350 colonies per plate as determined above in "Titering the transfected cells."
Spread the cells on rectangular plates [50ml of NZY agar containing ampicillin (750μl of a 100mg/ml stock solution per 500ml of agar medium, to yield 150μg/ml final concentration)] with IPTG and X-Gal as described in detail below. To each plate add:
35μl IPTG (0.1M)
70μl X-Gal (2% in DMF, which is 20mg/ml)
100μl transfected cells diluted to give 300 to 350 colonies/plate
Do not use fewer than 200μl total volume on each plate. If necessary, increase the volume to 200μl by adding the appropriate volume of LB-Amp.
Incubate overnight (16 – 18 hr) at 33°C. Then place at 4°C in darkness for 2 days to develop blue color before picking colonies.
Preparation of NZY-Amp agar plates:
For each 8 to 9 plates, melt 500ml of NZY agar in the microwave. Set the microwave to high power for 9 min (18 min for two bottles). Be certain to release the seal on the cap by turning counter-clockwise one full turn before turning on the microwave!
By careful visual inspection, and while swirling the contents, ensure that the agar is completely melted. Place the bottle(s) into an incubator at 55°C.
Allow the agar to equilibrate at 55°C, which takes several hours (unless it is an emergency in which case they can be largely pre-cooled with cool tap water prior to placing in the incubator).
If colonies are to be picked with the Gel-2-Well, the plates must be perfectly level. In this case use a 50ml graduated cylinder and pour plates in the Pure-Aire (white) sterile bench
Assuming (and ensure that this is truly the case) multiple bottles prepared at the same time are all safely at 55°C, then from one bottle pour a plate with 50ml of agar. This plate will be used as a control to ensure that the ampicillin is working as it should. If this assumption is not 100% safe, then prepare a control plate in this way from each bottle. Measure the 50ml with a sterile graduated cylinder.
To the bottle(s) from which 50ml of agar medium was removed add 675 μl of ampicillin stock at 100 mg/ml. To all other bottles add 750 μl of ampicillin. Mix well by swirling. Note that if the ampicillin is added when the medium is too warm, the ampicillin will be degraded and be unable to do its job.
Take one or two bottles at a time from the incubator and pour plates with 50ml of agar. Measure the agar medium with the 50ml graduated cylinder from step 5. Measure and pour carefully so that bubbles are minimized.
While the agar is still completely fluid, and as needed, pass the flame of a Bunsen burner very briefly over the surface to eliminate air bubbles. Note that it is not the heat but the CO2 that removes the bubbles, so do not heat the agar.
Allow the plates to cool and thus solidify uncovered in the sterile hood for 1 hr.
Cover with lids and store upside down. Normally they will be used on the day they are prepared. If to be used the next day, store overnight at 4°C.
Spreading SOLR cells on agar plates:
Prepare in advance a supply of autoclaved glass beads (6mm in diameter), 30 beads in each glass test tube.
To an agar plate add to three separate locations on the plate and in this order:
- 35μl IPTG(0.1M)
- 70μl X-Gal (2% in DMF)
- 100μl transfected SOLR cells at the appropriate dilution
- Without delay add glass beads from one of the tubes to the plate and ‘shake’ the plate by moving it around on the surface of the sterile bench. If appropriate get instructions from someone who already does it well. Generally it takes one to two minutes to complete the process. When the surface of the plate becomes dry there is a clear change in sound that you will come to recognize.
- The glass beads can be reused many times. When finished for the day, wash with 2% Micro in the wire strainer, rinse with tap water, then deionized water, then double distilled water. Set the beads aside to dry. Count 30 beads into glass tubes and autoclave.
- Cover and invert plates as they are. Just before 5 pm, place them in an incubator at 33°C. [If at 37°C colonies are too large in the morning.] Keeping plates inverted transfer to 4°C for blue color expression and storage until picked. They can be kept this way for a week or more.
Suggestions
The first day that MRF′ and SOLR cells are prepared in MgSO4 it can be very useful to use those cells in the evening to start new overnight cultures. In this way, if the cells prove to be successful, then they will be virtually certain to work well again the following day.
If good phagemids are known to exist (e.g., from a test for your ability to perform a mass excision effectively – see above), then include them as a positive control each time you do a transfection of SOLR cells.
It is also good practice to include a positive control for mass excision of unamplified library phage. Assuming you have already had good success performing a mass excision with amplified library phage, always include this excision as a positive control when you work with unamplified library phage.