Sam:LabNotes/Human/2009-10-29
Jump to navigation
Jump to search
Solexa sequencing library construction - real samples (human chro MDA product)
Background
- Sample information
- Sample No.10: 1/12.5 (200chr) 10hr incubation, ~36ng/uL in 20uL. (60ng/uL * 12 uL /20uL)
- Sample No.12: 1/25 (100chr) 10hr incubation, ~36ng/uL in 20uL. (60ng/uL * 12 uL /20uL)
- Only 10uL of each sample were used for this construction (save another 10uL sample as backup).
TBE-PAGE size selection
- TBE-PAGE condition: 5-well TBU. Run at 200V, 30min
- Sample: 10uL sample + 5uL H2O + 5uL 6X loading dye
- Ladder: 0.5uL 25bp ladder + 15uL H2O + 5uL 6X loading dye
- Loading: Low mass ladder -> No.10 -> No.12 -> 25bp ladder (Left to right)
- Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
- Cut the size 250+/-50bp (200bp~300bp)
File:Sam102909-TBE-size selection.JPG File:Sam102909-TBE-size selection-post cutting.JPG Left: Pre-cutting Right: Post-cutting
Blunting
- End-it Kit (EPICENTRE)
- Set reactions on cold block
1rxn 2.5rxn DNA 28.0 - 10X blunting buf. 4.0 10.0 dNTP mix 4.0 10.0 Blunting Enzyme 4.0 10.0 ------------------------------------ 40.0 30.0 30/2.5=12 --- DNA 28uL
- Mix the reaction by vortexing 5sec. Incubate at RT for 30min.
- During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (A-tailing)
- No inactivation needed, but purify the blunted DNA with QIAquick column. Elute in 30uL EB.
A-tailing
- Exo-Minus Klenow DNA polymerase(EPICENTRE)
- Set reactions on cold block
- Prepare the fresh dilution of 100mM dATP to 1mM dATP
1rxn 2.5rxns(EPI) DNA 30 - 10X buf. 4 10 (10X Knenow buf.) 1mM dATP 6 15 Klenow enzyme 2 5 ------------------------------------------ 40 30/2.5=12 - DNA 30uL
- Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.
- During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (Ligation)
- No inactivation needed, but purify the A-tailed DNA with QIAquick column. Elute in 12uL EB.
Ligation
- T4 ligation kit (Enzymatic)
- Set reactions on cold block
- Adaptor:Insert calculation
- Assume that only 25% DNA left after all of previous steps. 360ng*25% =90ng
- DNA before ligation: 90ng of 250b
- 90/(660x250)x10^6/1000 = 0.54uM --- Insert. Using 10:1~50:1 (Adaptor: Insert)will be 5.4uM ~ 27uM Y-adaptor needed.
- I decided to use 2uL of 20uM Y-adaptor which is even higher than 50:1 rule.
2.5rxn A-tailed DNA 11 - 2X rapid liagas buf. 15 37.5 20uM Y-adaptor 2 5.0 T4 ligase 2 5.0 --------------------------------- 30 47.5/2.5 = 19uL --- DNA 11 uL
- Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
- No inactivation needed, but purify the ligated DNA with QIAquick column. Elute in 10uL EB.
TBU-PAGE size selection
- TBU-PAGE condition: 5-well TBU. Run at 200V, 30min
- Sample: 10uL of ligation reaction + 10uL 2X TBU-loading dye
- Ladder: 0.5uL 25bp ladder + 9uL H2O + 10uL 2X TBU-loading dye
- Heat denaturing: 75C 15min -> 1min on cool block
- Loading: Low mass ladder -> No.10 -> No.12 -> 25bp ladder (Left to right)
- Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
File:Sam103009-TBU.jpg File:Sam103009-TBU-post cutting.jpg Left: Pre-cutting Right: Post-cutting
Size selected DNA recovery from TBU-PAGE
- Prepare gel shearing assemblies by placing a 0.5mL tube (punched a hole in bottom using G22 needle) in a 1.5-mL tube.
- Transfer the sliced gel in 0.5mL tubes. Centrifuge at 14000 rpm at RT for 3min.
- After centrifuge, remove the 0.5-mL tubes. Add 500uL 1X TE with the sheared gel in 1.5-mL tubes. Transfer the mixture to a fresh 1.5-mL tube.
- Incubate the gel-TE mixture at 37C for 1hr on the shaker (shaking at lowest speed)
- Centrifuge the gel-TE buffer mixture at 14000 rpm at RT for 1.5min. The gel debris will be pellet. Transfer the supernatant into a Nanosep column filter.
- Centrifuge the Nanosep columns at 14000 rpm at RT for 1.5min. The tiny gel debris will remain on the filter. Collect the flow through.
- Perform DNA purification with EtOH/NaOAc method.
- Sample + 2.5V 100% EtOH + 0.1V 3M NaOAc + 1.5uL Glycoblue.
- Gentaly invert the tube 20times. Incubate at -80C for 25min (or longer)
- Centrifuge at 4C, 10,000xg for 25min. Remove the supernatant(pour + 20p tips). Wash the pellet with 75% EtOH. Gentaly invert the tube 20times.
- Centrifuge at 4C, 10,000xg for 5min. Remove the supernatant(pour + 20p tips). Air dry the pellet in PCR hood (~10min).
- Elute the DNA in 20uL H2O.
qPCR amplification
- Only use helf of amount (10uL)for qPCR amplification.
- Comparing primers (primer from Alice vs. primers I ordered)
Master mix - Sam's primer mix - for L1, L3, L5, L7, N5 5.5 rxn H2O 35.6 195.8 Template (ligation product) 10.0 - Primer mix (f+r) (10uM) 4.0 22.0 SYBR 50X 0.4 2.2 2X iProof enzyme mix 50.0 275.0 --------------------------------------------- 100.0 495.0/5.5=90 --- 10uL template
Master mix - Alice's primers - for L2, L4, L6, L8, N6 5.5 rxn H2O 31.6 173.8 Template (ligation product) 10.0 - Primer f(10uM) 4.0 22.0 Primer r(10uM) 4.0 22.0 SYBR 50X 0.4 2.2 2X iProof enzyme mix 50.0 275.0 --------------------------------------------- 100.0 495.0/5.5=90 --- 10uL template
- Using the new program modifed based on Dr.Zhang's program (posted on 9-21-'09)
- Use 5 cycles for the first annealing temp(64).
- Use 16 cycles for the second annealing temp(72C).
Line1: 98C 30sec Line2: 98C 10sec Line3: 64C 20sec Line4: 72C 20sec Line5: Read plate Line6: Goto line 5 for 4 more times Line7: 98C 10sec Line8: 72C 20sec Line9: Read plate Line10: Goto line 7 for 15 more times Line11: 72C 5min Line12: 15C forever END
- Always terminate/pause the reaction at annealing step (72C).
- Usually the reaction will be close to saturation status around 7~8 cycles. Press the skip buttom to jump to final annealing step when it's closing to saturation stage.
- Check the qPCR product (3uL) on TBE gel to evaluate the library results.
File:Sam102709-TBE Solexa lib qPCR results-2.jpg
- The library sizes are as expected. The missed L6 sample was shown on the right size on N6 sample (The additional cutting for L6 sample)
- 270bp + Y-adaptor = 370bp; 130bp + Y-adaptor = 230bp
- The primer mix I made showed a stronger primer remains (~25bp) on the gel.
- Little smear were shown on L4 and L8 samples which may be caused by a over amplification.
Take home messages
- Elute DNA in EB after QIAquick washing is ok. (Not need to be eluted in H2O)
- Y-adaptor I made is working.
- All of the enzymatic reactions should be set up on cold block
- No inactivation step is needed in each reaction. Direct using QIAquick washing to terminate the enzymatic reaction.
- For the short insert (<200bp), the migration of ligation product on PAGE gel might be biased by the Y-adaptor's sturcture. Therefore a denatured PAGE (TBU) gel is recommanded for the size selection of ligation products. Using 5-well comb TBU is recommanded in order to prevent sample over-loading problem.
- Sample concentration is a key factor too. Use whole amount of product for any following step. The starting amount could be as low as 400ng if there is no size selection after sample shearing.