01/14/11
- ✓ Assemblies: KAH173, 174
- ✓ Order primers: for p65 and SP1 activation domain BioBricks
Assemblies
> Re-do KAH173 using dephos vector and max. insert
- KAH173: HPK/(E/S)/516 ✓ + KAH73/(E/X)/~5638 ✓
- KAH174: KAH75/(E/S)/558 ✓ + (1) KAH57/(E/X)/~4626
> Digests (Fermentas FD)
| Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
| DNA (plasmid) |
up to 25 μL
|
| 10x buffer |
3.0
|
| enzyme 1 |
1.0
|
| enzyme 2 |
1.0
|
| dH2O |
---
|
| |
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. KAH57 (E/X) |
0.506 |
1.81 |
25.3
|
> Dephosphorylation (Roche)
- KAH73(E/X)
| Reagent |
Volume
|
| DNA (clean digest) |
10.7 μL (500 ng)
|
| 10x buffer d.p. |
2.0
|
| phosphatase |
1.0
|
| dH2O |
6.3
|
| |
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|
> Ligations (1/15/11)
| Ligation |
Plate results (lig : neg crtl) 1/16/11
|
| 1. HPK(E/S)/516, 4.6 ng + KAH73(E/X)/~5638, 25 ng |
KAH173 >10:1 (Pick 2)
|
| 2. KAH73(E/X)/ 25 ng |
|
| 3. KAH75(E/S)/558, 6.0 ng + KAH57(E/X)/~4626, 25 ng |
KAH174 >10:1 (Pick 2)
|
| 4. KAH73(E/X)/ 25 ng |
|
| |
1 |
2 |
3 |
4
|
| Insert DNA |
3.0 |
--- |
1.0 |
---
|
| Vector DNA |
1.0 |
1.0 |
1.0 |
1.0
|
| 2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0
|
| dH2O |
--- |
3.0 |
2.0 |
3.0
|
| |
10 μL |
10 μL |
10 μL |
10 μL |
|
--> 10 min./ R.T.; add to 30 μL DH5α turbo
New BioBricks: p65, SP1 activation domains
--> Will PCR-clone these from human genomic DNA
> p65
- a.k.a. NFκB p65 subunit, RELA
- 261 amino acids from C-terminus (excluding stop codon) (Liu et al., 2001)
- Contains two adjacent PstI sites
- Primers:
- BB_p65 f1: 5'-CCTTTCTAGAGGAGCTGATCTGACT
- BB_p65 r1: 5'-AAGGCTGCAGCGGCCGCTACTAGTGATACAGACGATCGT
- mut_p65 1: 5'-ATCAAACTGCAaCTGCAaCAGGGCCTCTGA
--> Note (1/21/11): wrong ref sequence; redesign primers!
> SP1
- Has two activation domains that are sufficient for transcription at TATA/Inr promoters
- SP1AB is unable to activate a TATA-only promoter; will be interesting to see how this impacts global expression
- SP1A: aa's 83-262, 180 aa, 540 bp; 1 PstI site
- SP1B: aa's 263-542, 280 aa, 840 bp; 2 PstI sites
- SP1AB: aa's 83-621, 539 aa, 1617 bp; 3 PstI sites (clone & mutate this first, then use as template for A and B)
- Primers
- BB_SP1A f1: 5'-CCTTTCTAGAGGCCCGAGTCAGTCA
- BB_SP1A r1: 5'-AAGGCTGCAGCGGCCGCTACTAGTCCCATTCAGGGCCAC
- mut_SP1A 1: 5'-AACAGCTGCAaTTTGCTGCC
- BB_SP1B f1: 5'-CCTTTCTAGAAACATCACCTTGCTA
- BB_SP1B r1: 5'-AAGGCTGCAGCGGCCGCTACTAGTGATTCCTGAAGTACC
- mut_SP1B 1: 5'-CGTTTCTGCAaCTACCTTGA
- mut_SP1B 2: 5'-TACAGCTGCAaAACCTCCAA
- BB_SP1AB r1: 5'-AAGGCTGCAGCGGCCGCTACTAGTGCCAGGATCCCCCGA
|
Project name
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