Lab notebook
July 1st
Golden Gate Assembly of Phi29 Terminal protein (TP) ,Single stranded binding protein (SSBP) and Double Stranded binding protein (DSBP)+pGS21a backbone with Phi29 DNA Polymerase(DNAP)
- PCR amplify fragments
- rSAP digestion
- PaqCI digestion of insert + backbone
| Backbone | Insert |
|---|---|
| 5.71 uL DNA | 2.32 uL DNA |
| 5 uL Cutsmart Buffer | 5 uL Cutsmart Buffer |
| 1 uL PaqCI | 1 uL PaqCI |
| 0.25 uL PaqCI activator | 0.25 uL PaqCI activator |
| 38.1 uL H2O | 41.43 uL H2O |
Total : 50 uL
Incubate 37C for 1 hour , Cleanup elute 10 uL
Conc. Insert : 12.75 ng/uL
Conc. Backbone : 22.7 ng/uL
Ligation (3 tubes of equal volume / components):
| Tube 1 of 3 |
|---|
| 3.13 uL TPBP (insert) |
| 3.08 uL pGS21a (backbone) |
| 10 uL T7 Buffer |
| 2.79 uL H2O |
| 1 uL T7 Ligase |
Incubate 25C for 1 hr
Cleanup elute 10uL
Cleanup Assembly: | Concentration | | ------------- | | 11.65 ng/uL |
Make Agar+LB plates with Carbenicillin
July 2nd
Transform Phi29-pGS21a into DH5alpha using electroporation protocol
| Component | Volume |
|---|---|
| DNA | 2uL |
| DH5a | 25uL |
Plate 100uL on Carb
July 3rd
Run Colony PCR on 15 colonies to ensure insert length using DH5a as control
Create an overnight culture with 10mL LB+ scrape 1 colony with pipette tip and eject into test tube+ 10uL Carb
Let shake in 37°C for 12-16 hours overnight
July 4th
Miniprep overnight culture Elute: 30 uL | Colony | Qubit Concentration | | ---- | ---- | | 13 | 53.4 ng/uL | | 17 | 65.6 ng/uL|
Digest with KpnI+NdeI to confirm length
How the gel shoud look:
How the gel looked:
Dilute down to 30 ng/uL to send for sequence validation by SNPsaurus using m1v1=m2v2
send 10uL of diluted sample
July 5th
Sequence validated by SNPsaurus
Colony 17 perfect alignment match on benchling
Make overnight culture with T7+remix colony 17
July 6th
Miniprep Colony 17 for chemically competent transformation into T7
Elute 31 uL
Conc. : 80.4 ng/uL
Dilute to 40 ng/uL for transformation
1 uL plasmid, 50 uL cells
Serial Dilution using 1:10 ratio 3x
Plate 100uL on Carb
July 7th
Make an overnight culture with Colony plated on Carb and T7 cells with no colony (from stock)
July 8th
T7 OD600: 4.91
Col17 OD600: 4.62
Induce with 3 different concentrations of IPTG to test for protein toxicity on the cells by checking OD600 in a plate reader for 16 hours
T7 no plasmid and only LB used as controls
| Type of Cells | Concentration of IPTG | Volume of cells | Volume of IPTG | Volume of LB |
|---|---|---|---|---|
| Col 17 | 0mM | 8.65 uL | 0 uL | 791 uL |
| Col 17 | 0.01mM | 8.65 uL | 0.08 uL | 791 uL |
| Col 17 | 0.05mM | 8.65 uL | 0.4 uL | 790 uL |
| Col 17 | 0.1mM | 8.65 uL | 0.8 uL | 790 uL |
| Col 17 | 0.5mM | 8.65 uL | 4 uL | 787 uL |
| Col 17 | 1mM | 8.65 uL | 8 uL | 783 uL |
| T7 (-) | 0mM | 8.14 uL | 0 uL | 791 uL |
| T7 (-) | 0.1 mM | 8.14 uL | 0.8 uL | 790 uL |
| T7 (-) | 1mM | 8.14 uL | 8 uL | 783 uL |
| LB (-) | 0mM | 0 uL | 0 uL | 800 uL |
| LB (-) | 0.1mM | 0 uL | 0.8 uL | 799.2 uL |
| LB (-) | 1mM | 0 uL | 8 uL | 792 uL |
July 9th
Induction Curve:
Blue = pGS21a_Phi29_Colony17 in T7
Red = T7 no plasmid
Made more Linear Plasmid (pL) using PCR, digestion and ligation of fragments
| Fragment | Concentration after PCR eluted into 20uL |
|---|---|
| OriL | 83 ng/uL |
| Middle DHFR | 70.2 ng/uL |
| OriR | 85 ng/uL |
| Fragment | Concentration after Digest eluted into 12uL |
|---|---|
| OriL | 54.6 ng/uL |
| Middle DHFR | 65.4 ng/uL |
| OriR | 45 ng/uL |
Used Biomath Calculator to figure out volume of each fragment to add to ligation, 2:1 outer:inner
OriL: 212 bp-> 0.6ug -> 3.13 pmol-> 8uL
Middle DHFR: 348 bp -> 0.72 ug-> 3.13 pmol-> 11 uL
OriR: 216 bp-> 0.49ug-> 3.13 pmol-> 10uL
| pL Final Concentration eluted into 11uL |
|---|
| 58.8 ng/uL |
July 12th
Made plates:
10 ug/mL Carb, 0.5 mg/mL Tri, 0mM IPTG
10 ug/mL Carb, 0.5 mg/mL Tri, 0.1 mM IPTG
10 ug/mL Carb, 0.5 mg/mL Tri, 1 mM IPTG
Started 3 Large inductions in flasks using cells from overnight culture
3 Concentrations of IPTG: 0mM , 0.1mM, 1.0 mM
OD600 start: 0.05
OD600 end: 0.7
Make cells electrocompetent
Freeze some spun down cells in -80
Transform pL into T7 cells with pGS21a_Phi29_Col17 by Electroporation
Plate 100uL on compatible IPTG conc.
July 13th
No colonies found on any pL + pGS21a_Phi29 plates
Ran a protein gel using spun down cells from induction in -80
| Protein | Expected Size (kDa) |
|---|---|
| DNA Polymerase | 95 kDa |
| Terminal Protein | 31 kDa |
| Single Stranded Binding Protein | 13.3 kDa |
| Double Stranded Binding Protein | 12 kDa |
| Concentration of Supernatant| 0mM | 0.1mM | 1mM | T7 control | | ----| | 26.5 mg/mL | 13 mg/mL | 21.1 mg/mL | 1.8 mg/mL |
Ran 2 gels with 50 ug of each sample
| Volume ran on Gel | 0mM | 0.1mM | 1mM | T7 control | | ----| | 1.88 uL | 3.8 uL | 2.4 uL | 27 uL |
Stained one gel with Coomassie Blue, the other with Colloidal Overnight
July 14th
Imaged Both Gels, Gel stained with Colloidal was more clear
Gel stained with Colloidal:
Realized that DNAP size might be too hard to see on gel, ordered 7.5% Biorad protein gel to zoom in on 90 kDa
July 19th
Make more pL (non-thiolated and thiolated) using different primers
| Nonthio pL Conc. | Thio pL Conc. |
|---|---|
| 85.6 ng/uL | 68.8 ng/uL |
July 20th
Control for homemade electrocompetent cells/electroporation transformation efficency
Make more SEVA271 by transforming it into DH5a
Plate 100 uL on Kan diluted once with 1:10 dilution in LB
July 21st
Make overnight culture of SEVA271 colony
Make overnight culture of T7 pGS21a_Phi29_Col17 cells
Pour Kan+Carb plates
Order protein 1 gblock and primers from IDT to incorporate it into pGS21a_Phi29_Col17 backbone
July 23rd
Miniprep SEVA271 plasmid
Nick SEVA271 plasmid with Nb.BtsI
Make T7 pGS21a_Phi29_Col17 cells electrocompetent and transform with SEVA271
Plate 100 uL from serial dilution (3) onto Kan+Carb plates
July 26th
Count Colonies on SEVA271 plate and calculate transformation efficiency
Test for different concentrations of Trimethoprim using SEVA224+DHFR construct :
PCR amplify SEVA224 and DHFR using IDT primers
| Q5 | H2O | DNA | FWD Primer | REV Primer |
|---|---|---|---|---|
| 25 uL | 21 uL | 1 uL | 1.5 uL | 1.5 uL |
PCR cycling:
95C 1 min.
95C 15 sec
70C (for both primer sets )15 sec
72C 10 sec for DHFR, 50 sec for SEVA224
72C 5 min.
12C forever
Cleanup elute : 20uL each
| SEVA224 | DHFR |
|---|---|
| 70.9 ng/uL | 104.8 ng/uL |
Resuspend p1 gblock gene fragment in elution buffer:
- spin down in microcentrifuge 3-5 sec
- add 50 uL elution buffer
- vortex brifely
- incubate 50C for 20 min.
- vortex
- final conc: 8 ng/uL
- dilute to 1 ng/uL
July 27th
Made and ran gel of DHFR(2% gel) and SEVA24( 0.8% gel) PCR to ensure correct length
SEVA224 (~4000 bp shown next to 1 kb ladder) :
DHFR (~300bp shown next to 100 bp ladder) :
August 2nd
DpnI digest of SEVA224 backbone
| Cutsmart | DpnI | SEVA224 | H2O |
|---|---|---|---|
| 5 uL | 1 uL | 17 uL | 27 uL |
Incubate 37C for 1 hour
SEVA224 Conc. : 49.3 ng/uL
BsaI digest of SEVA224 and DHFR
| Cutsmart | BsaI | SEVA224 | H2O |
|---|---|---|---|
| 5 uL | 1 uL | 15 uL | 29 uL |
| Cutsmart | BsaI | DHFR | H2O |
|---|---|---|---|
| 5 uL | 1 uL | 16 uL | 28 uL |
30 min. in add 1 uL rSAP to SEVA224
Elute into 10 uL
| SEVA224 | DHFR |
|---|---|
| 37.9 ng/uL | 105.1 ng/uL |
Ligated fragments together with T4
| T4 Ligase Buffer | T4 Ligase | SEVA224 (0.12 pmol) | DHFR (0.36 pmol) | H2O |
|---|---|---|---|---|
| 2 uL | 1 uL | 8 uL | 0.7 uL | 8.3 uL |
Incubate: 16C 24 hours
65C 10 min.
10C forever
CLeanup Elute 8 uL
Conc.:
30.7 ng/uL
August 3rd
Chemically competent transformation into T7 Express ( transformed 1 uL DHFR_SEVA224 assembly)
Plated 100uL on Kan
Made plates with:
Kan + 0.5 mg/mL Tri
Kan + 0.25 mg/mL Tri
Kan + 0.1 mg/mL Tri
August 4th
Saw growth on Kan only selection plates
Restreaked 4 Colonies on each different concentration of Tri + Kan plates
August 5th
Saw growth on all Tri + Kan selection plates which means the concentration of Tri is not killing the cells
August 9th
PCR amplify P1 and pGS21a_Phi29 backbone
| Q5 | H2O | DNA | FWD Primer | REV Primer |
|---|---|---|---|---|
| 25 uL | 21 uL | 1 uL | 1.5 uL | 1.5 uL |
PCR cycling :
95C 1 min.
95C 15 sec
63C for P1, 70C for remix backbone) 15 sec
72C 10 sec for P1, 1 min. 30 sec for backbone
72C 5 min.
12C forever
Cleanup elute: 25uL
| P1 | pGS21a_Phi29 (Remix) |
|---|---|
| 101.45 ng/uL | 102.35 ng/uL |
Run a gel to confirm lengths before digesting
| Ladder | Remix | P1 |
|---|---|---|
| 3 uL dye | 3 uL dye | 3 uL dye |
| 1 uL 100 bp | 3 uL DNA | 3 uL DNA |
| 8 uL H2O | 6 uL H2O | 6 uL H2O |
P1 : 2% gel, 100 bp ladder, ~ 300 ng Remix : 0.8% gel, 1 kb ladder, ~ 300 ng
P1 gel (~300 bp ):
Remix gel ( ~ 9370 bp) :
- Primer dimerization shown at the bottom of the gel , remix was not used
August 10th
Make Overnight Culture of T7+remix cells for induction/ transformation
August 11th
OD600 of Overnight Culture : 3.17
Make Plates with 500 mL LB+Agar:
| 0 mM IPTG | 0.1 mM IPTG | 1 mM IPTG |
|---|---|---|
| 500 uL Carb | 500 uL Carb | 500 uL Carb |
| 0 uL IPTG | 500 uL IPTG | 5 mL IPTG |
| 5 mL Tri | 5 mL Tri | 5 mL Tri |
3 Induction Flasks:
| 0 mM IPTG | 0.1 mM IPTG | 1 mM IPTG |
|---|---|---|
| 50 mL LB | 50 mL LB | 50 mL LB |
| 0 uL IPTG | 50 uL IPTG | 500 uL IPTG |
| 789 uL cells | 789 uL Cells | 789 uL Cells |
Start : 0.05 OD600
End : 0.7 OD600
Make Induced Cells Electrocompetent
-
45 mL cells per tube (x uL H20 by 9)
-
Aliquot into 9 tubes
| 0 mM IPTG | 0.1 mM IPTG | 1 mM IPTG |
|---|---|---|
| no pL | no pL | no pL |
| pL | pL | pL |
| thiolated pL | thiolated pL | thiolated pL |
- Put 3 tubes in -80 to run protein gel on in the future
Transform with :
pL conc. : 85.6 ng/uL -> 0.4 uL
Thio pL conc. : 68.8 ng/uL -> 0.58 uL
Plated 100 uL on each conc.
August 12th
- No growth on any plate
Run gel on thiolated and non-thiolated pL to ensure ligated correctly
ran 300 ng DNA
| Ladder | Non-Thio pL | Thio pL |
|---|---|---|
| 3 uL dye | 3 uL dye | 3 uL dye |
| 1 uL 100 bp | 3.5 uL DNA | 4.4 uL DNA |
| 8 uL H2O | 5.5 uL H2O | 4.6 uL H2O |
1% gel , 125 V, 35 min.:
Correct length = 776 bp
August 13th
- To prevent primer self dimerization use smaller concentration of primers : 100 nmole
- pGS21a_phi29 backbone for P1
| Q5 | H2O | DNA | FWD Primer | REV Primer |
|---|---|---|---|---|
| 25 uL | 23.5 uL | 0.5 uL | 0.5 uL | 0.5 uL |
0.8% gel 130 V 35 min. :
- Still primer dimers
August 16th
Run protein gel on induced/ electrocompetent cells in -80
- use T7 cells as neg. control
- run on any kD gel and 7.5% gel to see DNAP
| T7 | 0 mM IPTG | 0.1 mM IPTG | 1.0 mM IPTG |
|---|---|---|---|
| 4.67 mg/mL | 56.6 mg/mL | 39.97 mg/mL | 31.15 mg/mL |
- dilute down to 5 mg/mL with Bugbuster
- added Laemli+BME to denature proteins
- ran 50 ug of each sample ( 10 uL)
200 V 50mA
initial voltage : 100V
rinse gel with DI H2O
stain overnight in colloidal coomassie stain
August 17th
Gel stained for 20 hours, broke in container and some slipped into aqueous waste
Rerun the any kD gel
| Protein | Expected Size (kDa) |
|---|---|
| DNA Polymerase | 95 kDa |
| Terminal Protein | 31 kDa |
| Single Stranded Binding Protein | 13.3 kDa |
| Double Stranded Binding Protein | 12 kDa |
Imaged both gels :
7.5% :
Ladder:
any kD :
Ladder :
- diluted original samples again with bugbuster
- ran new any kD and 7.5% gel
- 50 ug of each sample ( 10 uL of each sample )
- stained overnight with Coomassie Colloidal Stain for 18 hours
August 18th
- Try to use Betaine to remove primer dimer secondary structures from pGS21a_Remix_Backbone PCR reaction for adding P1
1M Betaine :
| Q5 | H2O | DNA | FWD Primer | REV Primer | Betaine |
|---|---|---|---|---|---|
| 25 uL | 11.5 uL | 0.5 uL | 1.5 uL | 1.5 uL | 10 uL |
1.35M Betaine :
| Q5 | H2O | DNA | FWD Primer | REV Primer | Betaine |
|---|---|---|---|---|---|
| 25 uL | 8 uL | 0.5 uL | 1.5 uL | 1.5 uL | 13.5 uL |
1.7M Betaine :
| Q5 | H2O | DNA | FWD Primer | REV Primer | Betaine |
|---|---|---|---|---|---|
| 25 uL | 4.5 uL | 0.5 uL | 1.5 uL | 1.5 uL | 17 uL |
Cleanup Elute 20 uL
Concentrations :
| 1M | 1.35 M | 1.7 M |
|---|---|---|
| 55.5 ng/uL | 34.1 ng/uL | 15.7 ng/uL |
0.8% gel, 140V, 30 min.
300 ng of each, 6 ng of miniprep product
Make overnight culture of T7+remix -10 mL LB -10uL Carb - scrape colony
August 19th
- Try using 1,2 propane-diol to get rid of dimers
- Try linearizing DNA (BsaI digest)
Miniprep T7+Remix overnight culture
Elute 35 uL
Conc. 1 : 11.95 ng/uL
Conc. 2 : 9.45 ng/uL
BsaI digest: - cut all of mp 1
| Cutsmart | BsaI-Hfv2 | H2O | DNA |
|---|---|---|---|
| 5 uL | 0.4 uL | 11.6 uL | 33 uL (400 ng) |
incubate 37C for 1 hour, heat inactivate enzymes 80C for 20 min
Cleanup elute 20 uL
Conc. 11.5 ng/uL
Remix PCR 1,2 Propane-diol ( not linearized ) :
| Q5 | H2O | DNA | FWD Primer | REV Primer | 1,2 Propane-diol |
|---|---|---|---|---|---|
| 25 uL | 18.5 uL | 0.5 uL | 1.5 uL | 1.5 uL | 3 uL |
BsaI cut Remix PCR 1,2 Propane-diol :
| Q5 | H2O | DNA | FWD Primer | REV Primer | 1,2 Propane-diol |
|---|---|---|---|---|---|
| 25 uL | 18.5 uL | 0.5 uL | 1.5 uL | 1.5 uL | 3 uL |
BsaI cut Remix PCR 1.35M Betaine :
| Q5 | H2O | DNA | FWD Primer | REV Primer | Betaine |
|---|---|---|---|---|---|
| 25 uL | 8 uL | 0.5 uL | 1.5 uL | 1.5 uL | 13.5 uL |
August 20th
Concentrations :
Remix PCR 1,2 Propane-diol ( not linearized ): 57.9 ng/uL
BsaI cut Remix PCR 1,2 Propane-diol : 84.6 ng/uL
BsaI cut Remix PCR 1.35M Betaine : 40.5 ng/uL
August 24th
How gel was oriented:
| PD remix PCR | BsaI cut Betaine remix PCR | BsaI cut PD remix PCR |
|---|---|---|
| 200 ng | 200 ng | 200 ng |
August 25th
- Make new assembly on Benchling where P1 is inserted after TP stop codon before DSBP RBS
- Make another new assembly on Benchling where P1 is inserted after DSBP stop codon before SSBP RBS
- Make new assembly on benchling for PCR mutagenesis of entire insert
- Make new assembly on benchling for PCR mutagenesis of TP+DNAP
-
Make new assembly on benchling for PCR mutagenesis of TP
-
Order all primers
September 1st
Make more non-Thio and Thio pL
Cleanup Elute 20 uL
Conc. :
| OriL | Middle | OriR |
|---|---|---|
| 120.45 ng/uL | 111.5 ng/uL | 138.9 ng/uL |
| Thio-OriL | Middle | Thio-OriR |
|---|---|---|
| 96.2 ng/uL | 124.05 ng/uL | 109.6 ng/uL |
September 2nd
BspQI digest w/ 19 uL DNA
| OriL | Middle | OriR |
|---|---|---|
| 19 uL DNA | 19 uL DNA | 19 uL DNA |
| 5 uL NEB 3.1 Buffer | 5 uL NEB 3.1 Buffer | 5 uL NEB 3.1 Buffer |
| 2.2 uL BspQI | 2.1 uL BspQI | 2.6 uL BspQI |
| 23.8 uL H2O | 23.9 uL H2O | 23.4 uL H2O |
| Thio-OriL | Middle | Thio-OriR |
|---|---|---|
| 19 uL DNA | 19 uL DNA | 19 uL DNA |
| 5 uL NEB 3.1 Buffer | 5 uL NEB 3.1 Buffer | 5 uL NEB 3.1 Buffer |
| 1.8 uL BspQI | 2.3 uL BspQI | 2 uL BspQI |
| 24.2 uL H2O | 23.7 uL H2O | 24 uL H2O |
Total: 50 uL
Incubate 50C for 1 hour
Cleanup elute 12 uL
Conc. :
| OriL | Middle | OriR |
|---|---|---|
| 99.3 ng/uL | 84.6 ng/uL | 85.3 ng/uL |
| Thio-OriL | Middle | Thio-OriR |
|---|---|---|
| 84.3 ng/uL | 106.5 ng/uL | 86.7 ng/uL |
1:2 backbone: Ori's ratio for ligation
3 PCR tubes all with same volume :
| T7 Ligase Buffer | OriL | Middle | OriR | H2O | T7 Ligase |
|---|---|---|---|---|---|
| 15 uL | 2.08 uL | 4 uL | 2.46 uL | 5.46 uL | 1 uL |
| T7 Ligase Buffer | Thio-OriL | Middle | Thio-OriR | H2O | T7 Ligase |
|---|---|---|---|---|---|
| 15 uL | 3.08 uL | 4 uL | 3.03 uL | 3.89 uL | 1 uL |
Total : 30 uL
Incubate 25C for 1 hour in thermocycler
Cleanup Elute 12 uL
Conc. :
pL : 55.75 ng/uL
Thio-pL : 47.1 ng/uL
September 3rd
- Amplify pGS21a_Remix backbone for p1 using 2 different sets of new primers ( one after DSBP stop codon the other after TP stop codon) but with same overhang as original assembly
- Add Betaine to ensure no primer dimers
| Q5 | H2O | DNA | FWD Primer | REV Primer | Betaine |
|---|---|---|---|---|---|
| 25 uL | 8 uL | 0.5 uL | 1.5 uL | 1.5 uL | 13.5 uL |
Cleanup Elute 20 uL
Concentration after TP : 30.8 ng/uL
Concentration after DSBP : 23.6 ng/uL
September 7th
Run gel: 0/8% gel, 130 V 30 min.
| Ladder | After DS | After Tp |
|---|---|---|
| 1 uL 1 kb Ladder | 6 uL DNA | 8.5 uL DNA |
| 3 uL dye | 3 uL dye | 3 uL dye |
| 10 uL H2O | 5 uL H2O | 2.5 uL H2O |
Gel ( want band around 9kb) :
- Run PCR on backbones of fragments that are going to be mutated :
- backbone for entire insert
- backbone for TPDNAP
- backbone for TP
How PCR was set up:
| Q5 | H2O | DNA | FWD Primer | REV Primer | Betaine |
|---|---|---|---|---|---|
| 25 uL | 8 uL | 0.5 uL | 1.5 uL | 1.5 uL | 13.5 uL |
September 8th
Cleanup elute 20 uL
Conc. :
Backbone for entire insert : 28.15 ng/uL
Backbone for TPDNAP : 76.2 ng/uL
Backbone for TP : 65.75 ng/uL
Run 300 ng on gel, 130 V, 30 min.
| Ladder | insert backbone | TPDNAP backbone | TP backbone |
|---|---|---|---|
| 1 uL 1 kb Ladder | 10.6 uL DNA | 3.93 uL DNA | 4.56 uL DNA |
| 3 uL dye | 3 uL dye | 3 uL dye | 3 uL dye |
| 10 uL H2O | 0.4 uL H2O | 7.1 uL H2O | 6.4 uL H2O |
Gel :
Make overnight culture of T7 and T7+remix cells
September 9th
Induction of T7+Remix cells
| Type of Cells | Concentration of IPTG | Volume of cells | Volume of IPTG | Volume of LB |
|---|---|---|---|---|
| T7+remix | 0mM | 12 uL | 0uL | 788 uL |
| T7+remix | 0.01mM | 12 uL | 0.08 uL | 787.9 uL |
| T7+remix | 0.05mM | 12 uL | 0.4 uL | 787.6 uL |
| T7+remix | 0.1mM | 12 uL | 0.8 uL | 787.2 uL |
| T7+remix | 0.5mM | 12 uL | 4 uL | 784 uL |
| T7+remix | 1mM | 12 uL | 8 uL | 780 uL |
| T7 (-) | 0mM | 11.1 uL | 0 uL | 788.9 uL |
| T7 (-) | 0.1 mM | 11.1 uL | 0.8 uL | 788.1 uL |
| T7 (-) | 1mM | 11.1 uL | 8 uL | 780.9 uL |
| LB (-) | 0mM | 0 uL | 0 uL | 800 uL |
| LB (-) | 0.1mM | 0 uL | 0.8 uL | 799.2 uL |
| LB (-) | 1mM | 0 uL | 8 uL | 792 uL |
Miniprep leftover overnight culture cells
Elute 50 uL
Conc: 33.45 ng/uL
Digest 30 uL miniprep remix DNA with KpnI-HF to linearize to try to make PCR run more smoothly
| Cutsmart | KpnI-Hfv2 | H2O | DNA |
|---|---|---|---|
| 5 uL | 1 uL | 14 uL | 30 uL (1000 ng) |
Cleanup elute 20 uL
Conc. : 27.4 ng/uL
-
Use new polymerase : Phusion
-
template DNA: whole insert backbone, TPDNAP backbone
| H2O | Phusion | FWD Primer | Rev Primer | DNA |
|---|---|---|---|---|
| 21.5 uL | 25 uL | 1.5 uL | 1.5 uL | 0.5 uL |
PCR Cycling :
98C 30 sec
98C 10 sec
68.3C for whole insert backbone (5347 bp), 67.8C for TPDNAP backbone (6684 bp) 15 sec
72C 2 min.
72C 10 min.
4C forever
September 10th
Cleanup Elute 21 uL
Conc. :
Insert backbone : 80.9 ng/uL
TPDNAP backbone : 195.6 ng/uL
0.8% gel, 130 V, 30 min., 210 ng insert backbone DNA, 1212 ng TPDNAP backbone (accidentally switched concentrations, meant to only run 500 ng)
| Ladder | insert backbone | TPDNAP backbone |
|---|---|---|
| 1 uL 1 kb Ladder | 2.6 uL DNA | 6.2 uL DNA |
| 3 uL dye | 3 uL dye | 3 uL dye |
| 8 uL H2O | 6.4 uL H2O | 2.8 uL H2O |
Gel:
- Ran again to ensure right band is not double band
- 500 ng each
| Ladder | insert backbone | TPDNAP backbone |
|---|---|---|
| 1 uL 1 kb Ladder | 6.2 uL DNA | 2.6 uL DNA |
| 3 uL dye | 3 uL dye | 3 uL dye |
| 8 uL H2O | 2.8 uL H2O | 6.4 uL H2O |
Redone gel:
September 13th
- Ran PCR on non-linearized mp remix for TP backbone for mutagenesis
- Used Fusion
- Annealing temp. : 66.2C
-
Extension time : 2 min. 40 sec ( ~ 8 kb )
-
Diluted mp product from 9/9 to 1 ng/uL
| H2O | Phusion | FWD Primer | Rev Primer | DNA |
|---|---|---|---|---|
| 21.5 uL | 25 uL | 1.5 uL | 1.5 uL | 0.5 uL |
Cleanup Elute 20 uL
Conc : 137.5 ng/uL
0.8% gel, 140 V , 30 min.
September 14th
Error Prone PCR Mutagenesis of Inserts Tubes : | Insert | Taq PCR Buffer | dNTP mix | 55 mM MgCl2 | 0.01 mM or 0.15 mM MnCl2 | FWD Primer | REV Primer | Taq Polymerase | Plasmid DNA | H2O | | ---- | ---- | ---- | ---- | ---- | ---- | ---- | ---- | ---- | ---- | | Whole Insert | 10 uL | 2 uL | 10 uL | 1 or 15 uL | 3 uL | 3 uL | 1 uL | 5 uL DNA (5 ng)| 65 or 51 uL H2O | | TPDNAP | 10 uL | 2 uL | 10 uL | 1 or 15 uL | 3 uL | 3 uL | 1 uL | 3 uL DNA (3 ng)| 67 or 53 uL H2O | | TP | 10 uL | 2 uL | 10 uL | 1 or 15 uL | 3 uL | 3 uL | 1 uL | 1.25 uL DNA (1.25 ng)| 68.75 or 54.75 uL H2O |
PCR: 95C 1 min.
94C 30 sec
(insert anneal : 58C, TPDNAP anneal : 55C, TP anneal : 58C ) 30 sec
72C 4 min. (insert), 2 min. (TPDNAP), 1 min. (TP)
20 cycles
72C 10 min.
4C forever
September 15th
Cleanup Elute 21 uL
Conc. :
Insert 0.01 mM : 88.7 ng/uL
Insert 0.15 mM : 118.8 ng/uL
TPDNAP 0.01 mM : 101.6 ng/uL
TPDNAP 0.15 mM : 110.3 ng/uL
TP 0.01 mM : 90.75 ng/uL
TP 0.15 mM : 114.9 ng/uL
Mix Equimolar Concentrations
| Insert 4043 bp | TPNAP 2706 bp | TP 764 bp |
|---|---|---|
| 0.63 pmol each | 1.08 pmol each | 3.42 pmol each |
| 19 uL 0.01 mM | 19 uL 0.01 mM | 19 uL 0.01 mM |
| 14.14 uL 0.15 mM | 17.5 uL 0.15 mM | 14.96 uL 0.15 mM |
Conc.:
Insert Mix : 101.2 ng/uL
TPDNAP Mix : 105.4 ng/uL
TP Mix : 102.4 ng/uL
1.2% gel, 130V, 36 min.
| 1 kb Ladder | Insert | TPDNAP | TP | 100 bp Ladder |
|---|---|---|---|---|
| 1 uL 1 kb Ladder | 2.96 uL DNA | 2.84 uL DNA | 2.93 uL DNA | 1 uL 100 bp Ladder |
| 3 uL dye | 3 uL dye | 3 uL dye | 3 uL dye | 3 uL dye |
| 8 uL H2O | 6.04 uL H2O | 6.16 uL H2O | 6.07 uL H2O | 8 uL H2O |
- lost TP/TP backbone to human error
DpnI Digest backbone and inserts:
| Cutsmart | DpnI | H2O | insert backbone DNA |
|---|---|---|---|
| 5 uL | 1 uL | 32.8 uL | 11.2 uL |
| Cutsmart | DpnI | H2O | insert DNA |
|---|---|---|---|
| 5 uL | 2 uL | 13 uL | 30 uL |
| Cutsmart | DpnI | H2O | TPDP backbone DNA |
|---|---|---|---|
| 5 uL | 1 uL | 32.8 uL | 11.2 uL |
| Cutsmart | DpnI | H2O | TPDP DNA |
|---|---|---|---|
| 5 uL | 2 uL | 10 uL | 33 uL |
Incubate 37C for 1 hour
Cleanup Elute 21 uL
Conc. :
Insert backbone : 35.4 ng/uL
Insert : 58.1 ng/uL
TPDP backbone : 54.9 ng/uL
TPDP : 76.7 ng/uL
September 20th
PaqCI Digest mutagens/ backbones
| Cutsmart | PaqCI | H2O | Insert backbone DNA | PaqCI activator |
|---|---|---|---|---|
| 5 uL | 1 uL | 23.75 uL | 20 uL | 0.25 uL |
| Cutsmart | PaqCI | H2O | Insert DNA | PaqCI activator |
|---|---|---|---|---|
| 5 uL | 1 uL | 23.75 uL | 20 uL | 0.25 uL |
| Cutsmart | PaqCI | H2O | TPDP backbone DNA | PaqCI activator |
|---|---|---|---|---|
| 5 uL | 1 uL | 23.75 uL | 20 uL | 0.25 uL |
| Cutsmart | PaqCI | H2O | TPDP DNA | PaqCI activator |
|---|---|---|---|---|
| 5 uL | 1 uL | 23.75 uL | 20 uL | 0.25 uL |
37C for 1 hour, 30 min. in add rSAP to backbones to prevent self-ligation, heat inactive 65C 20 min,
Cleanup Elute 21 uL
Conc:
Insert backbone : 19.4 ng/uL
Insert : 31.2 ng/uL
TPDP backbone : 26.4 ng/uL
TPDP : 45.8 ng/uL
Redo TP backbone PCR :
| H2O | Phusion | FWD Primer | Rev Primer | DNA |
|---|---|---|---|---|
| 21.5 uL | 25 uL | 1.5 uL | 1.5 uL | 0.5 uL |
Cleanup Elute 21 uL
Conc. : 158 ng/uL
PCR amplify TP with Agilent Genemorph error-prone PCR mutagenesis kit
| H2O | 10X Mutazyme II rxn buffer | 40 mM dNTP mix | FWD primer | REV primer | Mutazyme II DNAP | DNA |
|---|---|---|---|---|---|---|
| 41.5 uL | 5 uL | 1 uL | 0.25 uL | 0.25 uL | 1 uL | 1 uL |
Thermocycler conditions :
95C 2 min.
95C 30 sec
66C (Tm-5C) 30 sec
30 cycles
72C 1 min.
72C 10 min.
4C forever
September 21st
Cleanup Elute TP Mut 25 uL
Conc : 86.95 ng/uL
- PCR amplify p1 backbones ( after TP & after DSBP ) using Phusion
| H2O | Phusion | FWD Primer | Rev Primer | DNA |
|---|---|---|---|---|
| 21.5 uL | 25 uL | 1.5 uL | 1.5 uL | 0.5 uL |
PCR cycling :
98C 30 sec
98C 10 sec
72C 15 sec (for both after TP and after DSBP)
72C 3 min. (for both after TP and after DSBP)
72C 10 min.
4C forever
Cleanup Elute 25 uL
Conc. :
After TP : 147 ng/uL
After DS : 179 ng/uL
September 23rd
Run gel of Remix after TP and Remix afer DS to ensure Phusion PCR worked
0.8% gel , 300 ng DNA
| 1 kb Ladder | After TP | After DS |
|---|---|---|
| 1 uL 1 kb Ladder | 2 uL DNA | 1.7 uL DNA |
| 3 uL dye | 3 uL dye | 3 uL dye |
| 8 uL H2O | 7 uL H2O | 7.3 uL H2O |
DpnI digest of TP mut, Remix After TP, Remix After DS and Backbone for TP Mut
| Cutsmart | DpnI | H2O | TP Mut DNA |
|---|---|---|---|
| 5 uL | 2 uL | 19 uL | 24 uL |
| Cutsmart | DpnI | H2O | TP Mut backbone DNA |
|---|---|---|---|
| 5 uL | 2 uL | 23 uL | 20 uL |
| Cutsmart | DpnI | H2O | Remix for P1 after TP DNA |
|---|---|---|---|
| 5 uL | 2 uL | 20 uL | 23 uL |
| Cutsmart | DpnI | H2O | Remix for P1 after DS DNA |
|---|---|---|---|
| 5 uL | 2 uL | 20 uL | 23 uL |
Incubate 37C for 1 hour, heat inactivate 80C for 20 min.
Cleanup Elute 21 uL
Conc. :
TP Mut : 69.4 ng/uL
TP Mut backbone : 73.3 ng/uL
Remix after TP : 73.2 ng/uL
Remix after DS : 79.4 ng/uL
- Ligate Insert and TPDP mutagens to respective backbones
1:3 pmol ratio
insert backbone : 0.08 pmol, 280 ng DNA, 14.4 uL
insert : 0.24 pmol, 640 ng DNA, 20 uL
TPDP backbone : 0.12 pmol, 530 ng DNA, 20 uL
TPDP : 0.36 pmol, 640 ng DNA, 14 uL
3 tubes :
| T4 Buffer | T4 Ligase | H2O | Insert | Insert backbone |
|---|---|---|---|---|
| 2 uL | 1 uL | 5.54 uL | 6.66 uL | 5.54 uL |
3 tubes :
| T4 Buffer | T4 Ligase | H2O | TPDP | TPDP backbone |
|---|---|---|---|---|
| 2 uL | 1 uL | 5.71 uL | 4.63 uL | 6.66 uL |
Total : 20 uL
Incubation : 16C 24hr, 65C 10 min., 10C forever
Cleanup GGA Ligation Elute 15 uL
Conc. :
Whole Insert Mut : 28.1 ng/uL
TPDNAP Mut : 43.7 ng/uL
September 30th
PaqCI digest of : - TP Mut - TP Mut backbone - Remix for P1 after TP - Remix for P1 after DS - P1
| Cutsmart | PaqCI | H2O | TP Mut DNA | PaqCI activator |
|---|---|---|---|---|
| 5 uL | 1 uL | 23.75 uL | 20 uL | 0.25 uL |
| Cutsmart | PaqCI | H2O | TP Mut Backbone DNA | PaqCI activator |
|---|---|---|---|---|
| 5 uL | 1 uL | 23.75 uL | 20 uL | 0.25 uL |
| Cutsmart | PaqCI | H2O | Remix after TP DNA | PaqCI activator |
|---|---|---|---|---|
| 5 uL | 1 uL | 23.75 uL | 20 uL | 0.25 uL |
| Cutsmart | PaqCI | H2O | Remix after DS DNA | PaqCI activator |
|---|---|---|---|---|
| 5 uL | 1 uL | 23.75 uL | 20 uL | 0.25 uL |
| Cutsmart | PaqCI | H2O | P1 DNA | PaqCI activator |
|---|---|---|---|---|
| 5 uL | 1 uL | 21.75 uL | 22 uL | 0.25 uL |
Incubate 37 1 hour, 30 min. in add 1 uL rSAP to backbones to prevent self ligation, heat inactivate 65C 20 min.
Cleanup Elute 21 uL
Conc. :
TP Mut : 27.2 ng/uL
TP Mut backbone : 44.5 ng/uL
Remix after TP : 44.1 ng/uL
Remix after DS : 46.85 ng/uL
P1 : 50.1 ng/uL