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

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Line 77: Line 77:
| pSB1A3 || 2000 || 85.5 || 12.2 || 27.8
| pSB1A3 || 2000 || 85.5 || 12.2 || 27.8
|-
|-
-
| H2B || 378 || 40.4 || 4.9 ||  
+
| H2B || 378 || 40.4 || 4.9 || 35.1
|-
|-
-
| LOV+his || 450 || 18.6 || 12.0 || 28.0
+
| LOV+his || 450 || 18.6 || 12.6 || 27.4
|}
|}
Line 88: Line 88:
# H2B + LOV+his + pSB1A3
# H2B + LOV+his + pSB1A3
# pSB1A3
# pSB1A3
 +
# H2B + LOV+his + pSB1A3, 2xDNA
 +
# pSB1A3, 2xDNA
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
|- valign="top"
|- valign="top"
-
| Reagent || 1 || 2 || Master mix (x3)
+
| Reagent || 1 || 2 || Master mix (x3) || 3 || 4 || Master mix (x3)
|-
|-
-
| gg2 pSB1A3 || 1.0 || 1.0 || ---
+
| gg2 pSB1A3 || 1.0 || 1.0 || --- || 2.0 || 2.0 || ---
|-
|-
-
| gg3 LOV || 1.0 || H2O || ---
+
| gg3 LOV || 1.0 || H2O || --- || 2.0 || H2O || ---
|-
|-
-
| gg4 H2B+his || 1.0 || H2O || ---
+
| gg4 H2B+his || 1.0 || H2O || --- || 2.0 || H2O || ---
|-
|-
-
| 10x PRO ligase buffer || 1.0 || 1.0 || 3.0
+
| 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
+
| NEB T4 lgase || 0.25 || 0.25 || 0.75 || 0.25 || 0.25 || 0.75
|-
|-
-
| BsmBI || 0.5 || 0.5 || 1.5
+
| BsmBI || 0.5 || 0.5 || 1.5  || 0.5 || 0.5 || 1.5
|-
|-
-
| dH<sub>2</sub>O || 5.25 || 5.25 || 15.75  
+
| dH<sub>2</sub>O || 5.25 || 5.25 || 15.75 || 2.25 || 2.25 || 6.75  
|-
|-
-
| &nbsp; || 10.0 || 10.0
+
| &nbsp; || 10.0 || 10.0 ||  || 10.0 || 10.0 ||
|}
|}
* Aliquot 7.0 mix to each tube; add DNA/ H2O
* Aliquot 7.0 mix to each tube; add DNA/ H2O

Revision as of 15:23, 25 February 2013

Karmella's BioBrick Cloning Main project page
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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+his 0.04 1.667 40.4
2. LOV 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


(02/24/13)

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)
gg2 pSB1A3 1.0 1.0 --- 2.0 2.0 ---
gg3 LOV 1.0 H2O --- 2.0 H2O ---
gg4 H2B+his 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
  • Aliquot 7.0 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, ∞



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