04/22/15
- Rene - minipreps & sequencing: g53-2, g54
- LCR Development - assembly attempt #1
- Ryan - streak plates & liquid cultures (see 4/21/15)
Rene - minipreps & sequencing
- gRNA53-2
- gRNA54
- Sigma GenElute kit
- Elute w/ 75 μL elution sln.
- Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. g53-2 |
0.186 |
1.863 |
186.1
|
2. g54 |
0.205 |
1.895 |
205.2
|
- Submit to DNASU for sequencing
- g53-2, f (P139)
- g53-2, r (P139)
- g54, f (P139)
- g54, r (P139)
LCR - assembly attempt #1
Column-purify PCR fragments
- Use Qiagen PCR Clean-up, elute and back-elute w/ 30 μL elution buffer
- Gal4DB-mCh (4/22/15)
- ATF2 (4/22/15)
- MV10 (4/17/15)
Sample |
OD260 |
260/280 |
ng/μL
|
1. 5'p Gal4DB-mCh PCR |
0.139 |
1.825 |
138.6
|
2. 5'p ATF2 PCR |
0.191 |
1.828 |
190.8
|
2. 5'p MV10 PCR |
0.066 |
1.716 |
65.9
|
Dilute the purified dsDNA to 30 fmol/μL (30 nM)
- Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL
- Gal4DB-mCh (1170 bp) = 8.2
- ATF2 (906 bp) = 4.6
- MV10 (5191 bp) = 76.8 -- use 19.2 to make 25 uL of 15 fmol/μL sln., use 2x
- Add x to dH2O, final vol. = 50 μL
- Use 1.0 μL of diluted dsDNA per 10 μL LCR reaction
Prep the Oligo Bridges
- Note: Final conc. in LCR rxn. is 30 nM each
- Bring the IDT oligo pellet to 100μM with dH2O. nmoles oligo (on label) * 10 = μL H2O to add
- Make a 300 nM working solution (final volume = 100 μL) in a new tube. 3 μL of 100μM oligo stock + 97 μL dH2O = 100 μL
LCR Reactions (PCR fragments / oligo bridges)
- Gal4DB-mCh, ATF2, MV10 / LCRb_MV10_Gal4DB_rc, LCRb_mCh_ATF2_rc, LCRb_ATF2_MV10_rc
- Neg: MV10 / LCRb_MV10_Gal4DB_rc, LCRb_mCh_ATF2_rc, LCRb_ATF2_MV10_rc
- Note: use 5.0 μL of 15 fmol/μL MV10 instead of 2.5
Reagent |
Rxn1 |
Rxn2 (neg)
|
30 nM DNA (3 nM) |
10.0 |
5.0
|
300 nM Oligo Bridges (30 nM) |
7.5 |
7.5
|
10X Ampligase Buffer |
2.5 |
2.5
|
Ampligase |
1.0 |
1.0
|
dH2O |
4.0 |
6.5
|
|
25.0
|
Thermocycler: BioRad dual block
- 94 °C, 2 min.
- 50x[94°C, 10 sec; 55°C, 30 sec; 66°C, 60 sec
- 4 °C, ∞
Transformation
- Add to 60 μL DH5α-turbo; incubate 5 min/ice
- Plate on 100 μg/mL amp
- Grow at 37°C overnight
Ryan - Receiver cloning confirmations
Reagent
|
Volume
|
Expected: 1,2. AubR/MRV = ~3200, 886 3,4. BjaR/MRV = ~3200, 776 5,6. BraR/MRV = ~3200, 776 7,8. RpaR/MRV = ~3200, 779 MRV (empty) = ~3200
|
15 μL/lane; 1% agarose; Ladder
|
DNA(plasmid) |
3.0 μL
|
10X buffer |
1.5
|
EcoRI |
1.0
|
PstI |
1.0
|
dH2O |
8.5
|
|
15 μL --> 37°C/ ~15 min.
|
Conclusions
- AubR/MRV - success! Use clone 1
- BjaR/MRV - success! Use clone #1
|