Sam:LabNotes/Microbiome-new/2010-9-24
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Cloning Sanger sequencing test on E.coli gDNA MDA amplicons
Background
- To optimize the Sanger sequencing protocl for MDA amplicon validation
- This protocol also includes the S1 nucelase debranching and DNA Pol.I repairing treatments. I will compare the difference between with and without these treatments.
Plan
- Prepare 2ug of E.coli gDNA MDA amplicon for each reaction in 100uL volume
- S1 digestion(20units) for 0min(no S1 enzyme), 30min, 60min
- Aliquote 80uL of digested DNA for sonification into 1~2 kB (Keep another 20uL for comparison)
- Agarose gel size select the 1~2kB region. Recover DNA using Zymo kits or Montage filter.
Procedures
- Sample: Non-purified E.coli gDNA MDA amplicons made on 09-10-'10 (666ng/uL)
S1 nuclease digestion (debranching)
- Using prediluted S1 nuclease (10U/uL), USB brand.
- For digesting, 2ug MDA amplicon, 20U S1 nuclease will be used.
1rxn 6.5rxn DNA(666.ng/uL) 3.0 19.5 S1(10U/uL, USB) 2.0 - 2X S1 rxn buffer 50.0 325.0 H2O 45.0 292.5 ---------------------------------- 100.0 637.0 / 6.5 = 98 --- add S1 2uL or H2O 2uL
- Incubate at PCR thermocycler for 30min(2rxn) and 60min(2rxn).
- When incubation is completed, inactivate the reaction by adding 1uL 0.5M EDTA, vortex mixing, and incubating at 85C for 3min.
- Store the S1-treated DNA in -20C.
Sonification
- Following 30sec(on/off) protocol to shear DNA at 4C for 2min.
- Transfer 80uL of S1 treated DNA into the 1.7-mL tubes.
- NOTE: Use Bioexpress brand 1.7-mL tubes is recommanded. Using VWR 1.7mL tubes showed leaking problem after sonification.
Sonification program: Pulse on(30sec), Pulse off(30sec), Output power(5), Temp monitoring(No). Total time(2min).
- Store the sheared DNA in -20C
Agarose gel size selection (with Zymo Gel DNA recovery kit)(09-26-'10)
- reference link: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-4-16
- Prepae two reactions for each sample. Mix 25uL sample with 5uL 6X gel loading dye.
- Load 30uL samples into wells of 1% agarose gel made with 14-well comb using the long beak tips.
- Run the gel with 135V for 25min.
- Cut the DNA at the size of ~1kb.
Original gel File:Sam092710-precut of sheared Ecoli amplicons.jpg Post gel-cutting File:Sam092710-postcut of sheared Ecoli amplicons.jpg
- The cut gel slice (~100uL)was placed in 1.5-mL tube. Add 300uL ADB solution into tubes to dissolve the agarose gel.
- Incubate at 55C in hotplate for 10min (with 500 rpm agitation). Briefly vortexing to mix the dissolved gel.
- Transfer the melted agarose mixture onto Zymo-spin column in a collection tube. Centrifuge at 10,000xg for 30sec. Discard the flow-through.
- Add 200uL wash buffer (added EtOH into wash buffer first). Centrifuge at 10,000xg for 30sec. Discard the flow-through.
- Repeat the washing step again.
- Add 15uL pre-warmed clean water on the filter. Incubate at RT for 1min. Transfer the filter on a clean 1.5-mL tubes. Centrifuge at 10,000xg for 1min to elute the DNA.
Blunt(polishing) and Repairing Reactions (QUICK blunting kit, NEB)(DNA Pol.I, USB)(09-26-'10)
- Samples arrangement:
No.1 No.2 No.3 No.4 No.5 No.6 -------------------------------------------------------------------- S1 Exonuclease (2uL)(20U/uL,USB) 0m 0m 30m 30m 60m 60m DNA Pol.I(1uL)(10U/uL,USB) - yes - yes - yes --- adding 1uL
- Set up blunting reactions on the cold block:
1rxn 6.5rxn ----------------------------- H2O 4.0 26.00 10X buf 2.5 16.25 1mM dNTP 2.5 16.25 Blunt EZ(NEB) 1.0 6.50 Sample 15.0 - ----------------------------- 25.0 65.0/6.5 =10 --- Add DNA sample 15uL
- Briefly vortexing the blunting reaction: Incubate at RT for 30min
- Directly purify the Blunt reaction products using QIAquick without inactivation step.
- Mix 5Vol. PB buffer(125uL) with Blunted products(25uL) and then follow the purification procedures.
- Elute the purified DNA using 15uL prewarmed H2O.
Nanodrop results: File:Sam092610 nanodrop.jpg
QIAquick purification and Blunt ligation(09-27-’10)
- I am not sure if the blunted products could be used directly for ligation, so I purified both blunted reaction using QIAquick.
- In order to concentrate my samples, I pool the duplicate sample into the same tube before purification (ligation no1 + no2 => 50uL, ligation no.3 + no.4 => 50uL)
- Add another 50uL to make 100uL. Use 100uL as base volume for QIAquick purification.
- DNA were inlute in 20uL H2O.
- Nanodrop the concentration:
No.1 No.2 No.3 No.4 No.5 No.6 9.66 5.89 9.67 8.27 9.22 8.90 - ng/uL ------------------------------------------------ 1.55 2.54 1.55 1.81 1.62 1.68 - Amount need for 15ng Insert 5.45 4.46 5.45 5.19 5.38 5.32 - Amount of H2O needed to make up to 7uL
- Blunt-ligation (08-17-’10)
- Only 15ng of blunted DNA was used for blunt ligation.
- Original reference http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-7-10
No.1 No.2 No.3 No.4 No.5 No.6 --------------------------------------------------------------- Blunted-product(15ng) 1.55 2.54 1.55 1.81 1.62 1.68 H2O (fill up to 7uL) 5.45 4.46 5.45 5.19 5.38 5.32 1rxn 6.5rxn 10X Ligation buf. 1.00 6.50 pCR-Blunt (25ng/uL) 1.00 6.50 T4 DNA-ligase(4U/uL) 1.00 6.50 ------------------------------------------------ 3.00 19.50/6.5=3uL --- Transfer to each of reaction tubes that contained 7uL samples.
- Incubate at 16C for 1hr using PCR thermocycler. Perform transfection using the ligation product as soon as possible.
Heat-shock transfection
- Equilibrate water bath to 42C.
- Bring the SOC medium, LB plates (w/ antibiotics - Kanamycin 50ug/mL) to RT for 30min.
- Thaw the TOP10 E.coli competent cell vial on ice.
- Transfer 1uL ligation reactions into the vial of competent cells. Mix by gentle stiring using tips. (Mixing by repeat pipetting is prohibited in the protocol).
- Set up conpetent cell vials in the foam-board holder (for water bath). Incubate the vials on ice for 30 mins.
- Adjust the timer: set up 30sec and 2min.
- Heat shock the cells for 30 seconds at 42 C without shaking. Immediately transfer the vials on ice for 2min.
- Add 250ul of SOC medium into each vial (drop the SOC slution slowly throw the tube wall. No need for pipetting).
- Shake the vials horizontally at 37C for 1 hour using vortexor in the incubator at the lowest speed.
- Set up LB agar plates during the 1hr incubation.
- After 1hr shacking mixing, plate 50uL from each transformation vial on commercial LB agar plates (w/ Kanamycine, Teknova).
- There is no need of doing blue-white screening using X-gal, because the pBlunt plasmid has selfligation-proof design.
- Incubate plates at 37C for about 20hr.
- NOTE: I plated 12 plates in total. Two plates per sample. Incubation started from 2:00PM 09-27-'10.
Results (checked on 09-28-'10 12:00PM) Sample colonies count(white) NOTE ----------------------------------------- No.1-1 20 S1(0m) - No DNA Pol.I No.1-2 26 S1(0m) - No DNA Pol.I No.2-1 22 S1(0m) - W/ DNA Pol.I No.2-2 43 S1(0m) - W/ DNA Pol.I No.3-1 45 S1(30m) - No DNA Pol.I No.3-2 30 S1(30m) - No DNA Pol.I No.4-1 10 S1(30m) - W/ DNA Pol.I No.4-2 16 S1(30m) - W/ DNA Pol.I No.5-1 44 S1(60m) - No DNA Pol.I No.5-2 38 S1(60m) - No DNA Pol.I No.6-1 66 S1(60m) - W/ DNA Pol.I No.6-2 34 S1(60m) - W/ DNA Pol.I -----------------------------------------
- In general, the colonies number are fewer than I expected. It is possible that larger size insert is more difficult to clone. I will need to optimize the insert/vector ratio in the future.
- The current ligation protocol works great for short inserts (200~400bp). For long insert (1kB) I may need to decrease amount to half for the reaction. I am curious if using only 8ng inserts can improve the results.
- Except sample No.4, it looks like with S1 digestion treatment the successful cloning rate is a little bit increased.
- The S1 digestion removes the single strand DNA, which leaves dsDNA for more blunting ligating.
Cloning PCR using M13 primer (provided in Zero Blunt PCR cloning kit)(09-28-'10)
- The reason using M13 primer is due to confirm the existence of insert before sequencing. Besides, sequencing PCR products is cheaper than directed sequencing bacteria plasmid in Genewiz.
- M13 primer binding sites are flanking on the insert region. Additional margin size ~200bp.
- Since the M13 primer provided with kit are in 20uM, I will mix M13-f and M13-r together at 1:1 ratio to get the 10uM (f+r) mixture.
- Prepare 10uL H2O in PCR tube. Spike the colonies into H2O and stir a little bit.
- Incubate at 94C for 10min in thermocycler
- Using heat denaturing to lyse the cell and release the bacteria DNA. This approach is better then directly heat lyze the cell in PCR reaction(w/ enzyme) because the long-heating could damage the DNA polymerase as well.
- http://genome-tech.ucsd.edu/LabNotes/index.php/Dinh/NOTES/2010-7-3
- I picked up four colonies (only from the 1st duplicate plates) from each of different conditions (no.1~no.6). In total 4x6=24 colonies were picked for clonyl PCR reactions.
Taq-DNA polymerase PCR recipe 1rxn (8+1)x3=27rxn -------------------------------------- Template 2 - ------ heat denatured colonies M13 Primers (10uM, f+r) 1 27 H20 22 594 Taq 2x master Mix(NEB) 25 675 -------------------------------------- 50 1296/27=48 ---- 2uL Template Running at Program “PCR60” (Tm=60, 30cycle) 94C 3min -> (94C 30sec -> 60C 30sec -> 72C 1min) x 30cycle -> 72C 5min -> 15C forever
- Basically I use the standard PCR protocol with a longer extension time(1min)
- 10uL PCR products (leave 40uL for PCR product sequencing)were evaluated using 2% agasrose E-gel(run 12min)
Results: File:Sam092810-PCR validation no1-no4.jpg File:Sam092810-PCR validation no5-no6.jpg
- Most of colonies showed bands in expected size (1KB insert + 250bp vector margin).
- Still have a few colonies have no insert but only vector margin (~250bp)
- It is unknown why all samples in group 1 were failed.
- It seems like the longer S1 digestion (group 5 and group 6), the higher successful cloning rate.
- In order to submit equal number of sample for PCR sequencing from different groups. I did an additional Clonyl PCR using the unsuccessful groups.
New samples Soure plates: no.1 no.3 no.4 no.5 Sample number: 8 2 4 2 Taq-DNA polymerase PCR recipe 1rxn (8+1)x2=18rxn -------------------------------------- Template 2 - ------ heat denatured colonies M13 Primers (10uM, f+r) 1 18 H20 22 396 Taq 2x master Mix(NEB) 25 450 -------------------------------------- 50 864/18=48 ---- 2uL Template Running at Program “PCR60” (Tm=60, 30cycle) 94C 3min -> (94C 30sec -> 60C 30sec -> 72C 1min) x 30cycle -> 72C 5min -> 15C forever
Results: File:Sam092910-additional -1.jpg File:Sam092910-additional -2.jpg
AMpure beads purification (by multi-channel pipettor)
- Purify the PCR products (40uL) using AMpure beads with 1:1 (sample:beads) ratio.
- Set up multi-channel pipettor for purification.
- Pipettor Solution Basin - for 70% EtOH
- 8-well PCR tube strip - for RT-PCR H2O elution (30uL)x(3+1)=120uL/tube
- Procedures:
- Load 40uL libraries into PCR tube.
- Mixed libraries with 1V volume AMpure beads (40uL). Mix by repeat pipetting 15times (using multi-channel pipettor). Incubate at RT for 5min.
- Place the PCR tubes on magnetic board. Incubate at RT for 2min. Remove the supernatant(Remain the PCR tubes on magnetic board).
- Add 200uL 70% EtOH in to the PCR tube to rinse the AMpure bead (No pipetting). Incubate at RT for 30sec. Remove the supernatant.
- Repeat the EtOH wash again.
- Use 10p tips to remove the remained EtOH
- Air dry the the EtOH for 2min (Air dry more than 5min will dramatically reduce the DNA recovery rate).
- Remove the PCR tube away from magnetic board. Add 30uL RT-PCR grade H2O to elute the DNA.
- Mix the H2O and beads with repeat pipetting. Incubate at RT for 3min.
- Stay the PCR tube on the magnetic board. Incubate at RT for 2min. Transfer the supernatant (eluted DNA) into a fresh PCR tube strip.
- Nanodrop the purified DNA and stored it in -20C immediately. (NOTE: The remained beads in DNA solution will affect the Nanodrop results. It is suggested to leave sample tube strips on magnetic board when doing the Nanodrop measurment to avoid sucking beads)
Results: All sample showed normal curves with the concentration between 7~30ng/uL (in 30uL total volume) File:Sam092910 - nanodrop.jpg
- I will aliquote 50ng purified PCR products in 10uL H2O in PCR tubes strips, and send samples to Genewiz for PCR product sequencing.
- I will still use "pre-mixed" category for sending samples to save money, but I just need to prepare 10uL templates only since I am using universal primer M13F. Genewiz can add the M13F primer for free for me(confirmed).
Results
- Four sequences in each of six groups (conditions) were successfully sequenced using M13-primer.
- Raw sequence data were trimmed and BLAST to NCBI nt database.
- 23 out of 24 sequence showed no-gap matching to E.coli genome (but different strains). Sequences identity around 99~100%.
- The sequence 6-3 showed no result. I tried using BLASTX and the sequence was mapped to Agrobacterium tumefaciens, a member of Proteobacteria. However the alignment quality is poor (Identities = 28/118 (24%), Positives = 55/118 (47%), Gaps = 3/118 (2%))