Constructing Normalized cdna Libraries

Transcription

1 Constructing Normalized cdna Libraries The Center for Genomics and Bioinformatics Indiana University By: Zach Smith A. Introduction: In order to obtain high quality, low redundancy cdna libraries, it is necessary to employ some sort of normalization strategy to remove high abundance cdna transcripts. The following protocol was developed from the manuals of both the Evrogen TRIMMER-DIRECT cdna normalization kit (catalog number NK002) and the Clontech Creator SMART cdna Library Construction Kit (catalog number ). B. RNA isolation and quality control RNA isolation protocols will vary from organism to organism, so use an RNA isolation protocol appropriate to the organism in question. Remember, any cdna library can only be as good as the RNA it is built from, so high quality RNA is essential. Assay the RNA with the Agilent 2100 Bioanalyzer to assess RNA quantity and integrity. Quality RNA should have a 1.8 to 2.0 ratio between 28S and 18S rrna, and the gel-like image should show little to no RNA signal other than the ribosomal RNA. If the RNA is degraded, re-isolate new RNA and then proceed. Assess RNA purity and quantity on the Nanodrop before ss cdna synthesis. C. ss cdna Synthesis It is strongly recommended that everything that may potentially come into contact with your RNA samples be decontaminated with RNase ZAP in order to remove RNases and safeguard your RNA. 1. Denature the RNA for 5 minutes at 68 C in a thermocycler to remove any RNA secondary structure. 2. For each RNA sample, combine the following reagents in a.2ml PCR thermocycler tube:

2 Reagent Amount RNA sample (0.5 to 2.0 µg of 1-3µl RNA, use 1µl of the control RNA) CDS-3M adapter 1µl SMART IV oligonucleotide 1µl Sterile Water to 5µl Total Volume 5µl 3. Mix the contents and spin the tube briefly in a microcentrifuge. 4. Incubate the tube at 72 C for 2 minutes. 5. Incubate the tube on ice for 2 minutes. 6. Spin the tube briefly in a microcentrifuge to collect its contents at the bottom. 7. Add the following reagents to each reaction tube: Reagent Volume 5X First-Strand Buffer 2µl DTT (20uM) 1µl 50X dntp mix (10uM) 1µl Powerscript Reverse 1µl Transcriptase Total Volume in tube 10µl 8. Mix contents by gently pipetting and spin the tube briefly in a microcentrifuge. 9. Incubate the tube at 42 C for 1.5 hours in a thermocycler. 10. Place the tube on ice to terminate first-strand synthesis. Store the samples at - 20 C until the ds cdna synthesis step. ss cdna can be stored at -20 C for up to one month. D. ds cdna Synthesis 1. Preheat the thermocycler to 95 C. 2. Prepare the PCR Master Mix by combining the following reagent in a sterile 1.5ml tube per reaction:

3 Reagent Volume Sterile Water 80µl 10X Advantage 2 PCR 10µl Buffer 50X dntp mix 2µl 5 PCR Primer (Clontech 4µl kit) 50X Advantage 2 2µl Polymerase Mix Total Volume 98µl 2. Mix well by vortexing and spin the tubes briefly in a microcentrifuge. 3. Aliquot 98µl of the PCR Master Mix into each reaction tube. 4. Add 2µl of the ss cdna synthesis to the appropriate reaction tube and mix the contents by flicking the tube. Spin the tubes briefly in a microcentrifuge. 5. Commence thermal cycling using the following program on the Eppendorf Mastercycler. For the MJ tetrads, see the Evrogen manual for the MJ Research PCR Program: C for 2 minutes C for 7 seconds C for 30 seconds C for 6 minutes 5. Repeat to Step 2 for optimal cycles (see below) C for 4 minutes To determine the optimal number of PCR cycles for the amount of polya RNA used in the ss cdna synthesis: Total RNA (µg) polya RNA (µg) Number of PCR cycles It is essential to use the minimal number of PCR cycles when performing the ds CDNA synthesis to avoid the production of unspecific PCR product. It is better to undercycle than to overcycle, since undercycling can be rectified by placing the tubes back in the thermocycler. If the tube is overcycled the ds cdna synthesis must be done over again.

4 6. When the cycling is completed, analyze 6µlof each PCR product alongside the control and an appropriate DNA ladder on a 1.5% TAE agarose gel. ds cdna produced from poly(a)+ placenta RNA using SMART protocol. Ladder is a 1kb DNA ladder. Typical results include the following: A moderately strong smear of cdna ranging from 0.1 to 4kb, several bright bands corresponding to abundant transcripts, and some low-molecular-weight material. Source: Evrogen TRIMMER-DIRECT Manual Figure 7, page A PCR synthesis of about cycles is more than enough to make sure the rarest messages are present. 8. Purify the amplified ds cdna to remove primer excess, dntps, and salts using the Qiagen PCR Purification Kit (catalog number 28104). The only modification of the Qiagen PCR Purification Kit manual (p. 18) is to add 1µl of Sodium Acetate (3M, ph 4.8) provided in the Clontech kit to the sample after the 5X volume of PB buffer has been added in step 1. The Qiagen columns work on difference in ph to bind and then release the ds cdna, so by acidifying the ds cdna we increase the amount of ds cdna that binds the column and subsequently raise the elution yield. 9. Nanodrop the purified ds cdna to ascertain the concentration of the ds cdna. 10. Aliquot ng of ds cdna into a sterile 1.5 ml tube. Store the remaining purified ds cdna at -20 C. 11. OPTIONAL STEP: Bring the volume of your sample to 50µl by adding sterile water. (I have found that the extra volume allows the subsequent ethanol precipitation to work more effectively.) 12. Add 0.1 volumes of 3M Sodium Acetate, ph 4.8 to the reaction tube. 13. Add 2.5 volumes (sample volume + sodium acetate volume) of 100% ethanol to the reaction tube. 14. Vortex the mixture thoroughly and centrifuge the sample for minutes at 12,000-14,000 rpm at room temperature. 15. Carefully remove the supernatant. 16. Gently overlay the pellets with 100µl of 80% ethanol. 17. Centrifuge the tubes for 5 minutes at 12,000-14,000 rpm at room temperature. 18. Carefully remove the supernatant. 19. Repeat steps Air dry the pellet for minutes at room temperature. Be sure the pellet is completely dry before attempting to resuspend the samples.

5 21. Dissolve the pellet in sterile water to a final cdna concentration of about ng/µl 22. OPTIONAL STEP: Check the cdna quality and electrophorese 1µl of the cdna solution alongside 0.1ug of 1kb DNA size markers on a 1.5% TAE agarose gel. 23. This ds cdna can be stored at -20 C for up to three months prior to normalization. E. Normalization Before you start hybridization, make sure that the 4X Hybridization buffer has been allowed to stay at room temperature for at least minutes beforehand. If any pellet or white precipitate is present, warm the buffer to 37 C for 10 minutes to dissolve the pellet or precipitate. 1. For each sample to be normalized, combine the following reagents in a sterile 1.5 ml tube: Reagent Volume SMART-prepared ds cdna ( µl 1200ng) 4X Hybridization Buffer 4µl Sterile Water to 16µl Total 16µl 2. Mix contents and spin the tube briefly in a microcentrifuge. 3. Aliquot 4µl of the reaction mixture into each of the appropriately labeled tube (see table below). 4. Overlay the reaction with 3µl of mineral oil and centrifuge the sample at 14,000 rpm for 2 minutes. 5. Incubate the samples in the thermocycler at 68 C for 5 hours, then proceed immediately to the next step. During the 5 hour incubation step, its necessary to check the activity of the DSN enzyme. If the Normalization kit is new, dilute the DSN according to the following protocol: A. Add 25µl of DSN storage buffer to the lyophilized DSN enzyme. B. Mix the contents of the tube by gently flicking the tube. Spin the tube briefly in a microcentrifuge. Avoid foaming the mixture. C. Incubate the tube at room temperature for 5-7 minutes. D. Add 25µl of glycerol to the tube. E. Mix the contents of the tube by gently flicking the tube. Spin the tube briefly in a microcentrifuge. Avoid foaming the mixture. F. Store the DSN solution at -20 C.

6 To check DSN activity, follow the protocol below: A. Combine the following reagents in a sterile 1.5 ml tube: Reagent Volume Sterile Water 4µl DSN Control Template 4µl DSN Master Buffer 10µl Total Volume: 18µl B. Mix contents and spin the tube briefly in a microcentrifuge. C. Aliquot 9µl of the reaction mixture into to sterile PCR tubes labeled C (control) and E (experimental). D. Add 1µl of DSN storage buffer into the C-tube. Mix contents and spin the tube briefly in a microcentrifuge. E. Add 1µl of DSN solution (I generally use the lowest dilution of my DSN solution that I am using for a particular normalization, i.e. ¼ dilution) into the E-tube. Mix contents and spin the tube briefly in a microcentrifuge. F. Incubate the tubes in a thermal cycler at 65 C for 10 minutes. G. Add 10µl of DSN stop solution to each tube. Mix contents and spin the tube briefly in a microcentrifuge. Place the tubes at room temperature. H. Electrophorese 6µl of each reaction mixture on a 1.5% TAE agarose. The gel should look like the following gel: L DSN Activity Check L: DNA Ladder 1: Control 2: Unsuccessful 3: Successful Ladder is a 1kb DNA Ladder. Source: Evrogen TRIMMER-DIRECT Manual, Figure 6, page minutes before the 5 hour incubation period is over, pipette enough 2xDSN Master Buffer into a PCR thermocycler tube to suffice for each sample with an

7 extra 5µl for good measure. For example, for 12 tubes, pipette 65µl of DSN master buffer into the PCR tube. Place the tube into the thermocycler to preheat the DSN master buffer. 7. Make two dilutions of the DSN for the normalization, usually a ½ dilution of DSN and a ¼ dilution of DSN in 8. Pause the thermocycler and add 5µl of the preheated DSN master buffer to each of the normalization tubes. Do not remove the tubes from the thermocycler except for the time needed to add the DSN master buffer. 9. Incubate the tube at 68 C for 10 minutes. 10. Add the DSN enzyme as specified by the following table. Do not remove the tubes from the thermocycler. Source: Evrogen TRIMMER-DIRECT manual, Table 2, page Incubate the tubes in the thermal cycler at 68 C for 25 minutes. 12. Add 10µl of the DSN stop solution, mix contents and spin the tube briefly in a microcentrifuge. 13. Place the tubes on ice. 14. Add 20µl of sterile water to each tube. Mix contents and spin the tube briefly in a microcentrifuge. Place the tubes on ice. These normalized cdna samples can be stored at -20 C for two weeks. F. Post-DSN Amplifications For the first post-dsn amplification. 1. Preheat the thermocycler to 95 C. 2. Prepare a PCR master mix by adding the following reagents in the following order per tube: Reagent 1 X 4 X 8 X 12 X Sterile Water 40.5µl 162µl 324µl 486µl 10X Advantage 2 PCR buffer 5µl 20µl 40µl 60µl 50X dntp mix 1µl 4µl 8µl 12µl Evrogen M1 primer 1.5µl 6µl 12µl 18µl 50X Advantage 2 Polymerase Mix 1µl 4µl 8µl 12µl Total Volume 49µl 196µl 392µl 588µl

8 3. Mix well by vortexing. Mix contents and spin the tube briefly in a microcentrifuge. 4. Aliquot 1µl of each diluted cdna into an appropriately labeled sterile PCR tube. I have found it best to use a single tube for the control sample and a 3-tube strip for the experimental samples. 5. Aliquot 49µl of the PCR master mix into each of the reaction tubes. 6. Mix contents by gently flicking the tubes. Mix contents and spin the tube briefly in a microcentrifuge. 7. Commence thermal cycling on the Eppendorf Master Cycle with the following PCR program (Perkin-Elmer 480): C 2 minutes C 7 seconds C 30 seconds C 6 minutes Repeat to 2 13 cycles C 4 minutes 6. 4 C hold 8. Subject all tubes to 7 cycles (takes approximately 44 minutes on the Eppendorf Master Cycler. Label a 4 tube strip as follows: 7, 9, 11, and 13 for each control tube. 9. After 7 cycles are complete, place the experimental tubes at 4 C (I have found that the incubate function on the Eppendorf Master Cycler works best) and hold until the end of the experiment. 9A. Transfer 12µl of the 7 cycle PCR from the control tube to the strip tube labeled 7 9B. Run two additional PCR cycles (for a total of 9) with the remaining 38µl of PCR mix 9C. Transfer 12µl of the 9 cycle PCR from the control tube to the strip tube labeled 9. 9D. Run two additional PCR cycles (for a total of 11) with the remaining 26µl of PCR mix 9E. Transfer 12µl of the 11 cycle PCR from the control tube to the strip tube labeled 11. 9F. Run two additional PCR cycles (for a total of 13) with the remaining 14µl of PCR mix. 9G. Transfer 12µl of the 13 cycle PCR from the control tube to the strip tube labeled 13.

9 10. Electrophorese 6µl of each aliquot of each PCR reaction on a 1.5% TAE/agarose gel. The gel should look like: M Analysis for optimizing PCR parameters. M: Marker 7: 7 cycles 9: 9 cycles (optimal) 11: 11 cycles 13: 13 cycles 15: 15 cycles From the Evrogen TRIMMER-DIRECT Manual, Figure 9, page Determine the optimal number of cycles. This number is X. Use X to find the number of additional cycles, N, using the following formula: N=X-7. If the optimal number of cycles is 9, then N= 9-7 or Subject the experimental tubes to N+9 additional cycles (or 11 cycles from the example given in step 11). 13. When the cycling is complete electrophorese 6µl of each aliquot of each PCR reaction on a 1.5% TAE/agarose gel. Select the tubes with the most efficient normalization, which is illustrated in the following figure: M Analysis of cdna normalization results M: Marker 1: cdna from the control tube 2: cdna from the ¼ dilution DSN tube 3: cdna from the ½ dilution DSN tube 4: cdna from the 1µl DSN tube In this example, efficient normalization was achieved in lane 3, with the ½ dilution of DSN. Normalization was incomplete in lane 2, while in lane 4 DSN treatment was excessive, causing partial cdna degradation. From the Evrogen TRIMMER-DIRECT manual, Figure 10, page 30.

10 14. If the cdna from two or more tubes appears to be well normalized, combine the contents of these tubes into a single 1.5ml sterile tube, mix by vortexing, and spin the tubes briefly in a microcentrifuge. This amplified cdna can be stored at - 20 C for one month. For the Second Post-DSN amplification 1. Aliquot 2µl of the normalized cdna into a sterile 1.5ml tube, add 20µl of sterile water, mix well by vortexing and spin briefly in a microcentrifuge. 2. Aliquot 2µl of the control cdna into a sterile 1.5ml tube, add 20µl of sterile water, mix well by vortexing and spin briefly in a microcentrifuge. 3. Aliquot 2µl of the diluted normalized cdna into an appropriately labeled PCR tube. 4. Aliquot 2µl of the diluted control cdna into an appropriately labeled PCR tube. 5. Preheat the thermocycler to 95 C. 6. Prepare a PCR master mix by adding the following reagents in the following order per tube: Reagent 1X 2X 3X Sterile Water 80µl 160µl 240 µl 10X Advantage 2 PCR buffer 10µl 20µl 30 µl 50X dntp mix 2µl 4 µl 6 µl Evrogen Primer M2 4µl 8 µl 12 µl 50X Advantage 2 Polymerase mix 2µl 4 µl 6 µl Total Volume 98µl 196 µl 294 µl 7. Aliquot 98µl of the PCR master mix into each of the reaction tubes. 8. Mix contents by gently flicking the tubes. Mix contents and spin the tube briefly in a microcentrifuge. 9. Commence thermal cycling on the Eppendorf Master Cycler using the following PCR program (Perkins 480):

11 1. 95 C 2 minutes C 7 seconds C 30 seconds C 6 minutes 5. Repeat to cycles C 4 minutes 7. 4 C hold 10. When the cycling is complete electrophorese 6µl of each aliquot of each PCR reaction on a 1.5% TAE/agarose gel. The gel should be a smear of high molecular weight cdna. If there is no cdna product, low molecular weight material, or poor yield, see the Troubleshooting Guide in the Evrogen Manual (page 36). 11. The normalization procedure is now complete and the amplified normalized cdna can be stored at -20 C for one month. G. cdna Insert Processing This section contains three distinct phases: proteinasek digestion and cleanup, SFI I digestion, and size fractionation. ProteinaseK Digestion 1. In a sterile PCR tube, combine the 100µl reaction from the 2 nd Post-DSN amplification with 4µl of Proteins from the Clontech Creator SMART kit. Mix the contents and spin the tube briefly. 2. Incubate the tube in the thermocycler at 45 C for 30 minutes. 3. Purify the ds cdna to remove both ProteinaseK and any remaining Advantage 2 polymerase using the Qiagen PCR Purification Kit (catalog number 28104). The only modification of the Qiagen PCR Purification Kit manual (p. 18) is to add 1µl of Sodium Acetate (3M, ph 4.8) provided in the Clontech kit to the sample after the 5X volume of PB buffer has been added in step 1. The Qiagen columns work on difference in ph to bind and then release the ds cdna, so by acidifying the ds cdna we increase the amount of ds cdna that binds the column and subsequently raise the elution yield. 4. Nanodrop the purified ds cdna to ascertain the concentration of the ds cdna.

12 5. Determine the amount of purified ds cdna required to reach 2500ng of cdna in a 79µl solution using the following method: Concentration of purified ds cdna Volume of cdna needed for 2500ng 52.4 ng/µl 47.7µl 31.3µl Amount of water needed to reach 79µl SFI I Digestion 1. Take the 79µl solution of ds cdna from Step 5 above and combine it with the following reagents from the Clontech Creator SMART kit: Reagent Volume ds cdna 79µl 10X Sfi I Buffer 10µl Sfi I Enzyme 10µl 100X BSA 1µl Total 100µl 2. Mix well by gently flicking the tube or slow vortexing and spin down in a microcentrifuge. Incubate the tube at 50 C for 2 hours. 3. NOTE: If you are using the Chromaspin columns provided in the Clontech Creator SMART kit, add 2µl of 1% xylene cyanol to the tube above, mix well and place on ice until the column is prepared. Then follow the cdna size fractionation protocol found on page 26 of the Clontech manual. I have found the cdna Size Fractionation Columns from Invitrogen (catalog number ) to provide better and more consistent results because the bead mixture comes prepacked. The next stage of this protocol has been modified from the Appendix of the Invitrogen Cloneminer manual section entitled Size Fractionating Non- Radiolabeled cdna by Column Chromatography

13 cdna Size Fractionation 1. Before starting this procedure, make/obtain the following solutions: TEN buffer (10mM Tris-HCl, ph 7.5; 0.1mM EDTA; 25mM NaCl) 100% ethanol 7.5M NH 4 Oac 80% ethanol 2. Attach the column to a support stand, remove the top cap first, followed by the bottom cap. Allow the 20% ethanol solution to drain completely by gravity. 3. Once the column stop draining, pipette 0.8ml of TEN buffer into the column and allow it to drain completely. If the flow rate is noticeably slower than seconds, do not use the column. If the drop size from the column is not approximately 25-40µl, do not use the column. 4. Repeat the wash step three more times for a total of four washes and 3.2ml of TEN buffer. Let the column drain until dry. 5. Label 20 sterile 1.5ml tubes from 1 to 20. Place them in a rack under the bottom of the column with tube 1 directly beneath the column. 6. Add 50µl of TEN buffer to the SFI I digested cdna for a total volume of 150µl. Mix gently by pipetting. Add the entire sample to the column and let it drain into the resin bed. Collect the effluent into tube Move tube 2 under the column outlet and add 100µl of TEN buffer to the column. Collect the effluent into tube 2. Let the column drain completely. 8. Beginning with the next 100µl aliquot of TEN buffer, collect single-drop fractions into individual tubes starting with tube 3. Continue to add 100µl aliquots of TEN buffer until all 18 tubes (tubes 3-20) contain a single drop. 9. Electrophorese 6µl of each tube on a 1.5% TAE/agarose gel at 150 volts for 10 minutes. Collect the first three to four fractions in which cdna is visible in a sterile 1.5ml tube. These fractions will contain the largest molecular weight cdnas. 10. Add the following reagents to the tube of pooled cdna in this order: a) Glycogen (20 µg/µl) 1µl b) 7.5M NH 4 Oac 0.5 volume (0.5 x volume of cdna) c) 100% ethanol 2.5 volumes (2.5 x volume of cdna+nh 4 OAc) 11. Allow the cdna to precipitate at -20 C overnight. 12. Centrifuge the cdna at 4 C for 25 minutes at 14,000rpm

14 13. Carefully remove the supernatant while not disturbing the pellet. Add 150µl of 70% ethanol. 14. Centrifuge the sample at 4 C for 5 minutes at 14,000rpm. 15. Repeat steps 13 and Remove the 70% ethanol and allow the pellet to air-dry for minutes to completely remove the last traces of ethanol. 17. Resuspend the pellet in 7µl of Deionized Water and mix gently. Allow the sample to incubate at room temperature for 10 minutes. H. Ligation of ds cdna into pdnr-lib Be forewarned, I have found that the pdnr-lib vector provided in the Clontech Creator SMART kit is not completely dephophorylated, leading to recircularized vector that is chloramphenicol resistant but contains no cdna insert. At the very least, dephosphorylating the pdnr-lib vector should be done to lower the number of insertless clones using New England Biolabs Antarctic Phosphotase as described below. 1. Using 1µg of the pdnr-lib vector provided in the Clontech Creator SMART kit (10µl), set up the following reaction mix) in a sterile PCR tube. Reagent Volume pdnr-lib 10µl 10 X Antarctic Phosphotase Buffer 2µl Antarctic Phosphotase 1µl Total Volume 13µl 2. Mix the contents by gently flicking the tube and spin the tube in a microcentrifuge. 3. Incubate the sample at 37 C for 60 minutes. 4. Incubate the sample at 65 C for 10 minutes to inactivate the enzyme. 5. Proceed with the ligation.

15 Setting up the ligation reaction The Clontech Creator SMART kit recommends doing three different ligations for each sample using different amounts of cdna. I have found that the Ligation C mix works well for just about all the libraries I have made, so I recommend it in general. 1. Set up the following ligation mix (Ligation C): Reagent cdna ( ng/µl) pdnr-lib (0.1µg/µl) 10X Ligation Buffer ATP (10µM) T4 DNA Ligase Deionized Water Total Volume Volume 1.5µl 1.0µl 0.5µl 0.5µl 0.5µl 1.0µl 5.0µl 2. Incubate the sample at 16 C overnight. 3. Add 95µl of sterile DEPC-treated water to each sample. Transfer the resulting 100µl sample to a sterile 1.5ml tube. Add 1.5µl of glycogen. Mix with a pipette tip. Add 280µl of ice-cold 100% ethanol. Mix by gently rocking the tube. Place the tube at -80 C for 2.5 hours. 4. Centrifuge the samples at 15,000rpm for 25 minutes at 4 C. 5. Carefully remove the ethanol without disturbing the pellet. 6. Air dry the pellet for minutes at room temperature to remove every last trace of ethanol. 7. Resuspend the pellet in 5µl of sterile DEPC-treated water. Place the samples on ice. Transformation 1. While the samples are being spun down in step 4 above, place an appropriate number of electroporation cuvettes on ice. 2. In a sterile hood, pipette 970µl of Lb media into an appropriate number of 15ml Falcon tubes. Label each tube with the sample that will be added to it and place the tubes on ice. 3. When the Falcon tubes and cuvettes have chilled for ten minutes, retrieve the eletrocompetant cells from the -80 C freezer. Allow cells to thaw on ice. I have used electrocompetant TOP10 cells from Invitrogen (catalog number C )

16 for this step, though Clontech recommends using DH5α prepared according to their instructions on page 44 of the Clontech Creator SMART manual. I have achieved good results with the TOP10 cells, but I have yet to attempt using the Clontech DH5α cells, so I cannot venture an opinion on them. For the Electrocompetant TOP10 cells from Invitrogen, each tube contains 50µl of cells, which is enough for two transformations. Once they have thawed these cells cannot be refrozen. 4. Add 25µl of thawed cells to the 5µl of resuspended recombinant plasmids. Mix thoroughly with a pipette tip. 5. Transfer the cell/plasmid mixture to a chilled electroporation cuvette. 6. Electroporate by discharging. The time constant for a good transformation should range between 4.6 and 5.0. Any reading lower or higher than this range should be treated with suspicion. 7. Using a microcapillary tip, transfer the transformants from the electroporation cuvette to the appropriate Falcon tube. Be careful not to pipette the sample overmuch. Mix the transformants into the LB by gently flicking the tube. Place the Falcon tube back on ice until all transformations are complete. 8. Incubate the transformants at 37 C for 1 hour, shaking at 225rpm. Titering Plasmid Libraries 1. Before this procedure, make a suitable number of LB/Chloramphenicol (LB/CAM from this point on) plates. This is done by first making and sterilizing LB solid media and allowing it to cool to about 60 C. Then add the chloramphenicol from a previously produced stock (30mg/ml in 100% ethanol, 1000X) to the media before pouring into 100mm plates. For this step, I use a 50ml Falcon tube to measure out the media, and then I add 50µl of chloramphenicol, mix, and pour 25ml each into two plates. If the plates were stored at 4 C, allow to warm to room temperature before use. 2. Remove 1µl of the library and add it to 1ml of LB broth in a sterile 1.5ml tube. Mix by gentle vortexing. This is Dilution A (1:10 3 ). 3. Remove 1µl of Dilution A and add it to 1ml of LB broth in a sterile 1.5ml tube. Mix by gentle vortexing. This is Dilution B (1:10 6 ). 4. Add 50µl of LB to a 1.5ml tube labeled DilA. Pipette 1µl of Dilution A into this 50µl aliquot and spread the entire sample on a prewarmed LB/CAM plate. 5. Remove 50µl and 100µl aliquots from Dilution B and spread them onto separate pre-warmed LB/CAM plates. 6. Leave the plates at room temperature for minutes to allow the inoculums to soak into the agar. Store the libraries at 4 C. 7. Invert the plates and allow them to incubate overnight at 37 C. 8. Count the colonies to determine the titer using the following formulas Colony number Dilution A x 10 3 x 10 3 = cfu/ml

17 (Colony number Dilution B/plating volume) x 10 3 x 10 3 x 10 3 = cfu/ml I. Small Scale Quality Control 1. Label 2 8-tube PCR strips 1-16 for each library produced. Pipette 50µl of sterile water into each tube. 2. Pick 16 colonies at random from the titer plates and add each colony to a separate tube. Seal the strips and vortex until the colony is no longer visible. Spin the strips briefly in a microcentrifuge. 3. Incubate the samples at 98 C for 6-8 minutes in a thermocycler to lyse the cells. 4. Spin the strips at 5700rpm for 6 minutes to pellet the cell debris. 5. After allowing the reagents to thaw on ice, create a PCR master mix according to the table below. For this step I have use the Eppendorf Taq DNA polymerase kit (Eppendorf catalog number ) with good results. The PCR primers for this amplification are as follows: pdnrm13fw: GTG TAA AAC GAC GGC CAG TAG pdnrm13rev: AAA CAG CTA TGA CCA TGT TCA C Reagent 1X 16X Sterile Water 4.3µl 68.8µl 10X Taq Polymerase Buffer 1µl 16µl 10mM dntp mix 0.2µl 3.2µl 10µM Forward PCR Primer 0.2µl 3.2µl 10µM Reverse PCR Primer 0.2µl 3.2µl Taq DNA Polymerase 0.1µl 1.6µl Template 4µl - Total Volume 10µl 6µl each 6. Label a new set of 2 8-tube PCR strips 1 to Aliquot 6µl of the PCR master mix into each of the 16 tubes. 8. Add 4µl of the sample plasmid DNA into the appropriate tube containing the PCR master mix. Be careful not to disturb the pelleted cell debris while doing so. 9. Seal the PCR strips containing the PCR reaction, mix by gentle vortexing, and spin briefly in a microcentrifuge. Pre-heat the thermocycler to 95 C.

18 10. Begin thermal cycling of the samples on the Eppendorf Master Cycler using the following PCR program: C 1 minute C 30 seconds C 30 seconds C 4 minutes 5. Repeat to 2 22X C 5 minutes 7. 4 C hold 11. When thermal cycling is complete, electrophorese the samples on a 1.5% TAE/agarose gel with a 1kb ladder (I use Promega s 1kb DNA Ladder catalog number G5711). The resulting gel should look like this: 12. If there is no cdna insert present in the plasmid, this primer set will produce a 500bp product. If the gel shows a large number of 500bp bands (more than 4) or if it has a uniform line of 500bp bands, the library is no good and you will have to go back to at least the ligation step if not further. If your gel looks like the one above, proceed to large scale quality control. J. Large Scale Quality Control Before this beginning this procedure, produce mm LB/CAM plates for colony picking each library. See step 1 in the Titering plasmid libraries section. I use a 50ml Falcon tube to measure out the media, and then I add 50µl of chloramphenicol, mix, and pour the entire tube into a single 150mm Petri dish. 1. Remove the libraries from the 4 C freezer and allow them to warm to room temperature. Place the liquid LB media and the large plates at room temperature as well.

19 2. Label two 1.5ml tubes 1µl and 2µl for each library. Add 400µl of LB media to the tube labeled 1µl and add 100µl of LB to the tube labeled 2µl. 3. Add 4µl of the library to the tube marked 1µl, so that the there is 1µl of library per 100µl of LB. This solution is enough to make 4 colony picking plates. 4. Add 2µl of the library to the tube marked 2µl. This solution is enough to make 1 colony plate at double the concentration of the colony plates listed above in step Take the solution from step 3 and plate 101µl of the solution on each of 4 150mm LB/CAM plates. 6. Take the solution from step 4 and plate the entire solution on a single 150mm LB/CAM plate. 7. Allow the plates to sit for 10 minutes to allow the inoculums to soak into the plate. 8. Invert the plates and incubate them overnight at 37 C. Restore the libraries to the 4 C freezer. 9. If the number of colonies is satisfactory, remove the libraries from the 4 C freezer and allow them to come to room temperature. Label 2 sterile 1.5ml tubes and divide the 1ml stock into 2 500µl stocks. Add 200µl of 80% glycerol to each tube and mix via gentle pipetting. Freeze the tubes by placing them at -80 C. The libraries are now safe. 10. Place 2 boxes of flat toothpicks (round toothpicks tend to absorb media more efficiently than flat toothpicks) into a glass beaker; cover it with aluminum foil, and autoclave. These will be needed for colony picking. 11. Make 1L of Freezing Buffer according to the recipe below: 1M stock solutions 1M K 2 HPO 4 1M KH 2 PO 4 1M Na-citrate 1M MgSO 4 1M (NH 4 ) 2 SO 4 Recipe 28.5g K 2 HPO 4 in 125ml water 17g KH 2 PO 4 in 125ml water 36.8g Na-citrate in 125ml water 15.1g MgSO 4 in 125ml water 16.5g (NH 4 ) 2 SO 4 in 125ml water

20 Freezing Buffer Recipe Reagent 1M K 2 HPO 4 1M KH 2 PO 4 1M Na-citrate 1M MgSO 4 1M (NH 4 ) 2 SO 4 Glycerol (4.4% total volume) NaCl Tryptone Yeast Extract Water Total Volume Amount 36ml 13.2ml 1.7ml 0.4ml 6.8ml 44ml 10g 10g 5g to 1L 1L 12. Autoclave the Freezing Buffer and allow it to cool to room temperature. 13. For each library, use the following steps to produce a 384well QC plate a) Measure out 30ml of Freezing Buffer with a 50ml Falcon tube. Add 30µl of chloramphenicol to the Freezing Buffer and mix. Aliquot 60µl of the Freezing Buffer/chloramphenicol mix to each well on a 384-well plate (from Nunc). We thankfully have the Bio-mek for this task. b) Using an autoclaved toothpick, pick a single colony from one of the colony picking plates and place it in well A1. Pick colonies for the first 6 columns of the 384-well plate, mark the stopping place with a Sharpie, and then remove the toothpicks. This is to keep the toothpicks from absorbing the Freezing Buffer. Repeat this process until each well has been inoculated. c) Place the 384-well plate at 37 C overnight. d) Wrap the colony picking plates in parafilm and store at 4 C. 14. Produce and autoclave 1L of 2xYT media according to the following recipe: In 900ml of Deionized water, add 16g of tryptone, 10g of yeast extract, and 5g of NaCl. Adjust the ph to 7.0 with NaOH. Adjust the volume to 1L using Deionized water. Autoclave and allow it to cool to room temperature. 15. For each 384-well plate, we will inoculate 1 96-deepwell plate for plasmid preps. To produce a 96-deepwell plate, use the following steps.

21 a) Measure out 150ml of 2xYT media 50ml at a time using a 50ml Falcon tube. Add 50µl of chloramphenicol to each 50ml tube of 2xYT media. Mix well and add to a reservoir. b) Using a multichannel pipettor, pipette 1.4ml of 2xYT/chloramphenicol mix into each well of the 96-deepwell plate. c) Using the Bio-mek, take a 5µl inoculum from the 384-well plate and inoculate the 96-deepwell plate. Incubate the 96-deepwell plate at 37 C overnight at 225rpm. Seal the 384-well plate with a foil plate seal and store the 384-well plate at -80 C. 16. After allowing the 96-deepwell plate to incubate overnight, plasmid prep the 96 samples using the Perfectprep Plasmid 96 Vac, Direct Bind from Eppendorf to prep the plasmids. The only modifications made to this protocol are to substitute Solutions 1, 2, and 3 from the Eppendorf Perfectprep kit for Buffers P1, P2, and P3 from Qiagen s Plasmid Purification Handbook (page 68). To make these buffers, follow the recipes below: Buffer P1 Dissolve 6.06g Tris base, 3.72g Na 2 EDTA-2H 2 O in 800ml of water. Adjust the ph to 8.0 with HCl. Adjust the volume to 1L with water. Add 100mg RNase A per liter P1 Buffer P2 Dissolve 8.0g NaOH pellets in 950ml water, 50ml 20% SDS solution. Adjust the volume to 1L with water. Buffer P3 Dissolve 294.5g potassium acetate in 500ml water. Adjust the ph to 5.5 with glacial acetic acid (~110ml). Adjust the volume to 1L with water. 17. After the samples have been plasmid prepped, set up the following PCR master mix: Reagent 1X 100X Sterile Water 4.3µl 430µl 10X Taq Polymerase Buffer 1µl 100µl 10mM dntp mix 0.2µl 20µl 10µM Forward PCR Primer 0.2µl 20µl 10µM Reverse PCR Primer 0.2µl 20µl Taq DNA Polymerase 0.1µl 10µl Template 4µl - Total Volume 10µl 6µl each

22 18. Label a 96-well PCR plate 19. Aliquot 6µl of the PCR master mix into each well of the 96-well PCR plate. 20. Pipette 4µl of each sample into the appropriate well of the 96-well PCR plate. 21. Seal the 96-well PCR plate with a thermo seal and vortex gently to mix. 22. Spin the 96-well PCR plate at 1000rpm for 1 minute to collect the reactions at the bottom of the plate. 23. Preheat the thermocycler to 95 C. 24. Begin thermal cycling the samples in the Eppendorf Master Cycler according to the PCR program found in Step 10 of the Small Scale Quality Control section. 25. When thermal cycling is complete, electrophorese the samples on a 1.5% TAE/agarose gel with a 1kb ladder. The resulting gel should look similar to this: 26. If the gel appears satisfactory, i.e. the samples range from about 700bp to 2Kb and there are not a large number of 500bp samples, use the Kodak Image station to assess the average lengths of the inserts and proceed to sequencing. If not, you ll have to go back at least to the ligation step. 27. For sequencing the 96 sample QC, set up the following sequencing reaction mix according to the table below. The sequencing primers are as follows: pdnrlib30-50fwd: TAT ACG AAG TTA TCA GTC GAC G M13rev: AAA CAG CTA TGA CCA TGT TCA C Reagent 1X 100X 5X BigDye Buffer 2µl 200µl 2µM pdnr-libfwd 2µl 200µl Primer BigDye 1µl 100µl Template 5µl - Total Volume 10µl 5µl each

23 28. Label a 96-well PCR plate. 29. Aliquot 5µl of the sequencing master mix into each well of the 96-well sequencing plate. 30. Pipette 5µl of each sample plasmid prep into the appropriate well of the 96-well sequencing plate. 31. Seal the 96-well PCR plate with a thermo seal and vortex gently to mix. 32. Spin the 96-well PCR plate at 1000rpm for 1 minute to collect the reactions at the bottom of the plate. 33. Preheat the thermocycler to 96 C. 34. Begin thermal cycling according to the following PCR program: C 30 seconds C 30 seconds C 15 seconds C 4 minutes 5. Repeat to 2 27X 6. 4 C hold 35. Sequencing is done in the IMBI DNA facility by Lawrence Washington. 36. Analyze the sequencing results using the CGB EST Pipeline, graciously provided by Haixu Tang. The pipeline is currently under construction.