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='''Cloning Sanger sequencing test on E.coli gDNA MDA amplicons'''=
='''Cloning Sanger sequencing test on E.coli gDNA MDA amplicons (protocol validation)'''=


==Background==
==Background==
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*Using prediluted S1 nuclease (10U/uL), USB brand.
*Using prediluted S1 nuclease (10U/uL), USB brand.
*For digesting, 2ug MDA amplicon, 20U S1 nuclease will be used.
*For digesting, 2ug MDA amplicon, 20U S1 nuclease will be used.
**In the previous test, 1ug DNA digested with 10U S1 for 30min digestion didn't no structure changes compared to 10min digestion.
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-9-7
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-9-7
                      1rxn      6.5rxn
                                  1rxn      6.5rxn
   DNA(666.ng/uL)      3.0      19.5
   2ug MDA product(666.ng/uL)      3.0      19.5
   S1(10U/uL, USB)     2.0      -
   S1(10U/uL, USB)                 2.0      -
   2X S1 rxn buffer   50.0    325.0
   2X S1 rxn buffer(Sam made)      50.0    325.0
   H2O                 45.0    292.5
   H2O                             45.0    292.5
   ----------------------------------
   -------------------------------------------------
                    100.0    637.0 / 6.5 = 98 --- add S1 2uL or H2O 2uL
                                100.0    637.0 / 6.5 = 98 --- add S1 2uL or H2O 2uL


*Incubate at PCR thermocycler for 30min(2rxn) and 60min(2rxn).
*Incubate at 37C using 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.
*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.
*Store the S1-treated DNA in -20C.


===Sonification===
===Sonification===
*Following 30sec(on/off) protocol to shear DNA at 4C for 2min.
*Following 30sec(on/off) protocol to shear DNA at 4C for 2min (To get fragments size ~1kb).
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-9-9
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-9-9
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-9-10
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-9-10
*Transfer 80uL of S1 treated DNA into the 1.7-mL tubes.
*Transfer 80uL of S1 treated DNA into the 1.7-mL tubes (Keep another 20uL (400ng) for non-shearing comparison)
**'''NOTE: Use Bioexpress brand 1.7-mL tubes is recommanded. Using VWR 1.7mL tubes showed leaking problem after sonification.'''
**'''NOTE: Use Bioexpress brand 1.7-mL tubes is recommanded. Using VWR 1.7mL tubes showed leaking problem after sonification.'''
  Sonification program:
  Sonification program:
Line 44: Line 45:
===Agarose gel size selection (with Zymo Gel DNA recovery kit)(09-26-'10)===
===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
*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.  
*'''Prepare two reactions for each sample'''. Mix 25uL sample (500ng) 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.
*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.
*Run the gel with 135V for 25min.
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*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.
*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 reactions (QUICK blunting kit, NEB)(09-26-'10)===
===Blunting(polishing) and Repairing reactions (QUICK blunting kit, NEB)(DNA Pol.I, USB)(09-26-'10)===
*Samples arrangemetns:
*Samples arrangement:
                      No.1  No.2  No.3  No.4  No.5  No.6
                                  No.1  No.2  No.3  No.4  No.5  No.6
  -------------------------------------------------------
  --------------------------------------------------------------------
  S1 treatment        0m    0m    30m  30m  60m  60m
  S1 Exonuclease (2uL)(20U/uL,USB)  0m    0m    30m  30m  60m  60m
  Pol.I treatment(1uL) -     yes  -    yes  -    yes
  DNA Pol.I(1uL)(10U/uL,USB)          -   yes  -    yes  -    yes --- adding 1uL


*Set up blunting reactions on the cold block:
*Set up blunting reactions on the cold block:
Line 77: Line 78:
  Sample      15.0      -
  Sample      15.0      -
  -----------------------------
  -----------------------------
               25.0      65.0/6.5 =10 --- Add DNA sample 15uL
               25.0      65.0/6.5 =10 --- Add DNA sample 15uL  
*Briefly vortexing the blunting reaction: Incubate at RT for 30min
*Briefly vortexing the blunting reaction: Incubate at RT for 30min
*For reactions need to add DNA Pol.I, directly add it on the top regardless the changes of total volume.
*Directly purify the Blunt reaction products using QIAquick without inactivation step.
*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.
**Mix 5Vol. PB buffer(125uL) with Blunted products(25uL) and then follow the purification procedures.
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  Nanodrop results:
  Nanodrop results:
  [[File:sam092610_nanodrop.jpg|800px]]
  [[File:sam092610_nanodrop.jpg|800px]]
*DNA recovery rate calculation:
**Loading 500ng (2000ng/100x25)sheared DNA on each of wells for size selection.
**Size select the 1kb regions and Zymo-column purified -> Quick blunt -> QIAquick purified -> Eluted in 15uL H2O.
**Total recovered amount ~150ng. 150/500 = ~1/3
**'''About 1/3 of DNA recovered from loaded DNA in gel.'''. Given that I only cut a partial region (1/2~1/3) of sheared DNA for recovery, the recovery rate is actually very good.


===QIAquick purification and Blunt ligation(09-27-’10)===
===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.
*Only 15ng of blunted DNA was used for blunt ligation.
*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)
*Original reference http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-7-10
*Add another 50uL to make 100uL. Use 100uL as base volume for QIAquick purification.
Prepared sample in 15ng and add H2O to 7uL
*DNA were inlute in 20uL H2O.
*Nanodrop the concentration:
   No.1  No.2  No.3  No.4  No.5  No.6
   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
   9.66  5.89  9.67  8.27  9.22  8.90 - ng/uL
Line 96: Line 102:
   1.55  2.54  1.55  1.81  1.62  1.68 - Amount need for 15ng Insert
   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
   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)
Preparing Blunt-ligation master-mix
**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
                         1rxn    6.5rxn
  10X Ligation buf.      1.00    6.50       
  10X Ligation buf.      1.00    6.50       
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*Shake the vials horizontally at 37C for 1 hour using vortexor in the incubator at the lowest speed.
*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.
*Set up LB agar plates during the 1hr incubation.
*After 1hr shacking mixing, plate '''50uL''' from each transformation vial on agar plates (w/ Kanamycine).  
*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.
**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.
*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.
*'''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)
  Results (checked on 09-28-'10 12:00PM)
  Sample    colonies number(white)
  --------------------------------
  Sample    colonies count(white) NOTE
  No.1-1      20  
  -----------------------------------------
  No.1-2      26
  No.1-1      20   S1(0m)  - No DNA Pol.I
  No.2-1      22
  No.1-2      26   S1(0m)  - No DNA Pol.I
  No.2-2      43
  No.2-1      22   S1(0m)  - W/ DNA Pol.I
  No.3-1      45
  No.2-2      43   S1(0m)  - W/ DNA Pol.I 
  No.3-2      30
  No.3-1      45   S1(30m) - No DNA Pol.I
  No.4-1      10
  No.3-2      30   S1(30m) - No DNA Pol.I
  No.4-2      16
  No.4-1      10   S1(30m) - W/ DNA Pol.I
  No.5-1      44
  No.4-2      16   S1(30m) - W/ DNA Pol.I
  No.5-2      38
  No.5-1      44   S1(60m) - No DNA Pol.I
  No.6-1      66
  No.5-2      38   S1(60m) - No DNA Pol.I
  No.6-2      34
  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)===
===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.
*The reason using M13 primer is 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.
**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.
**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.
Line 155: Line 159:
**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.
**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
**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
  Taq-DNA polymerase PCR recipe
                           1rxn   27rxn
                           1rxn   (8+1)x3=27rxn
  --------------------------------------
  --------------------------------------
  Template                2      -    ------ heat denatured colonies
  Template                2      -    ------ heat denatured colonies
  M13 Primers (10uM, f+r)  1      27   
  M13 Primers (10uM, f+r)  1      27   
  H20                    22      594
  H20                    22      594
  Taq 2x Maxter Mix       25      675
  Taq 2x master Mix(NEB)  25      675
  --------------------------------------
  --------------------------------------
                         50    1296/27=48 ---- 2uL Template
                         50    1296/27=48 ---- 2uL Template
   
   
  Running at Program “PCR60” (Tm=60, 30cycle)
  Running at Program “PCR60” (Tm=60, 30cycle)
  94C 3min -> (94C 30sec -> 60C 20sec -> 72C 20sec ) x 30cycle -> 72C 5min -> 15C forever
  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|500px]] [[File:sam092810-PCR validation no5-no6.jpg|500px]]
*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|500px]] [[File:sam092910-additional -2.jpg|500px]]
 
===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|800px]]
*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%.[[Media:Genewiz 093010 - E.coli MDA Sanger sequencing - NCBI Blast bblast nt.pdf|BATCH BLAST results (PDF)]]
**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 for this sequence (Identities = 24%, Gaps = 2%)

Latest revision as of 17:08, 11 January 2011

Cloning Sanger sequencing test on E.coli gDNA MDA amplicons (protocol validation)[edit]

Background[edit]

  • 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[edit]

  • 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[edit]

S1 nuclease digestion (debranching)[edit]

                                  1rxn      6.5rxn
 2ug MDA product(666.ng/uL)       3.0      19.5
 S1(10U/uL, USB)                  2.0       -
 2X S1 rxn buffer(Sam made)      50.0     325.0
 H2O                             45.0     292.5
 -------------------------------------------------
                                100.0     637.0 / 6.5 = 98 --- add S1 2uL or H2O 2uL
  • Incubate at 37C using 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[edit]

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)[edit]

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.

Blunting(polishing) and Repairing reactions (QUICK blunting kit, NEB)(DNA Pol.I, USB)(09-26-'10)[edit]

  • 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
  • For reactions need to add DNA Pol.I, directly add it on the top regardless the changes of total volume.
  • 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
  • DNA recovery rate calculation:
    • Loading 500ng (2000ng/100x25)sheared DNA on each of wells for size selection.
    • Size select the 1kb regions and Zymo-column purified -> Quick blunt -> QIAquick purified -> Eluted in 15uL H2O.
    • Total recovered amount ~150ng. 150/500 = ~1/3
    • About 1/3 of DNA recovered from loaded DNA in gel.. Given that I only cut a partial region (1/2~1/3) of sheared DNA for recovery, the recovery rate is actually very good.

Blunt ligation(09-27-’10)[edit]

Prepared sample in 15ng and add H2O to 7uL

  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
Preparing Blunt-ligation master-mix
   
                        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[edit]

  • 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)[edit]

  • The reason using M13 primer is 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
  • 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)[edit]

  • 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[edit]

  • 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%.BATCH BLAST results (PDF)
    • 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 for this sequence (Identities = 24%, Gaps = 2%)