User:Karmella Haynes/Notebook/BioBrick cloning/2013/02/23

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(→02/23/13)
(→02/23/13)
Line 95: Line 95:
| Reagent || 1 || 2 || Master mix (x3) || 3 || 4 || Master mix (x3)
| Reagent || 1 || 2 || Master mix (x3) || 3 || 4 || Master mix (x3)
|-
|-
-
| gg2 pSB1A3 || 1.0 || 1.0 || --- || 2.0 || 2.0 || ---
+
| pSB1A3 gg || 1.0 || 1.0 || --- || 2.0 || 2.0 || ---
|-
|-
-
| gg3 H2B || 1.0 || H2O || --- || 2.0 || H2O || ---
+
| H2B gg || 1.0 || H2O || --- || 2.0 || H2O || ---
|-
|-
-
| gg4 LOV+his || 1.0 || H2O || --- || 2.0 || H2O || ---
+
| LOV+his gg || 1.0 || H2O || --- || 2.0 || H2O || ---
|-
|-
| 10x PRO ligase buffer || 1.0 || 1.0 || 3.0 || 1.0 || 1.0 || 3.0
| 10x PRO ligase buffer || 1.0 || 1.0 || 3.0 || 1.0 || 1.0 || 3.0
Line 111: Line 111:
|   || 10.0 || 10.0 ||  || 10.0 || 10.0 ||  
|   || 10.0 || 10.0 ||  || 10.0 || 10.0 ||  
|}
|}
-
* Aliquot 7.0 mix to each tube; add DNA/ H2O
+
* For 1 and 2, aliquot 7.0 mix to each tube; add DNA/ H2O
 +
* For 3 and 4, aliquot 4.0 mix to each tube; add DNA/ H2O
Thermal cycling
Thermal cycling

Revision as of 16:22, 25 February 2013

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

02/23/13

  • Type IIS Assembly, PCR: H2B-LOV+his


Type IIS Assembly, PCR: LOV-H2B+his

  1. H2B = 378 bp, 1A3/H2B fwd + H2B/LOV rev (Template: H2B-GFP from Addgene)
  2. LOV = 429 bp, LOV fwd + LOV+his/1A3 rev (Template: LOV from Addgene); note: reverse primer adds a 6-his tag and TAA stop (21 bp)
  3. pSB1A3 = ~2000, gg0001 v2 + gg0002 v2
  • Be sure to use the H2B and LOV primers ordered on 2/4/2003. The names are redundant with a previous version.

PCR

  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞
Reagent Volume Expected:
1. H2B = 378
2. LOV = 450
3, 4. pSB1A3 = ~2000 		Hover name
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.2 μL
primer 1 1.0
primer 2 1.0
2x GoTaq 25.0
dH2O 22.8
  50.0 μL


Purification

  • Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
  • Measure [DNA]
Sample OD 260 260/280 ng/μL
1. H2B 0.04 1.667 40.4
2. LOV+his 0.019 1.562 18.6
3. pSB1A3 0.069 1.754 69.4
4. pSB1A3 0.085 1.817 85.5


Dilutions: to final concentration of 20 fmol/μL (40 μL)

  • x = length in bp ÷ measured ng/μL * 0.013 * 40
DNA length (bp) ng/μL Vol. (x) + dH2O
pSB1A3 2000 85.5 12.2 27.8
H2B 378 40.4 4.9 35.1
LOV+his 450 18.6 12.6 27.4


(02/25/13)

Golden Gate Reactions

  1. H2B + LOV+his + pSB1A3
  2. pSB1A3
  3. H2B + LOV+his + pSB1A3, 2xDNA
  4. pSB1A3, 2xDNA
Reagent 1 2 Master mix (x3) 3 4 Master mix (x3)
pSB1A3 gg 1.0 1.0 --- 2.0 2.0 ---
H2B gg 1.0 H2O --- 2.0 H2O ---
LOV+his gg 1.0 H2O --- 2.0 H2O ---
10x PRO ligase buffer 1.0 1.0 3.0 1.0 1.0 3.0
NEB T4 lgase 0.25 0.25 0.75 0.25 0.25 0.75
BsmBI 0.5 0.5 1.5 0.5 0.5 1.5
dH2O 5.25 5.25 15.75 2.25 2.25 6.75
  10.0 10.0 10.0 10.0
  • For 1 and 2, aliquot 7.0 mix to each tube; add DNA/ H2O
  • For 3 and 4, aliquot 4.0 mix to each tube; add DNA/ H2O

Thermal cycling

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.
  • 4°C, ∞



Retrieved from "http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/02/23"
Personal tools