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

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'''Type IIS Assembly, PCR: LOV-H2B+his'''
'''Type IIS Assembly, PCR: LOV-H2B+his'''
# H2B = 378 bp, 1A3/H2B fwd + H2B/LOV rev (Template: H2B-GFP from Addgene)
# H2B = 378 bp, 1A3/H2B fwd + H2B/LOV rev (Template: H2B-GFP from Addgene)
-
# LOV = 429 bp, LOV fwd + LOV+his/1A3 rev (Template: LOV from Addgene); note: reverse primer adds a 6-his tag (18 bp)
+
# 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)
# pSB1A3 = ~2000, gg0001 v2 + gg0002 v2
# 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.
* Be sure to use the H2B and LOV primers ordered on 2/4/2003. The names are redundant with a previous version.
Line 28: Line 28:
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
-
| rowspan="7" | <u>Expected:</u><br>1. H2B = 378<br>2. LOV = 447<br>3, 4. pSB1A3 = ~2000
+
| rowspan="7" | <u>Expected:</u><br>1. H2B = 378<br>2. LOV = 450<br>3, 4. pSB1A3 = ~2000
| rowspan="7" | [[Image:KAH022313_gel1.jpg|200px|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan="7" | [[Image:KAH022313_gel1.jpg|200px|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA(plasmid) || 0.2 μL
| DNA(plasmid) || 0.2 μL
|-
|-
-
| primer 1 || 1.0
+
| 10 μM primer 1 || 1.0
|-
|-
-
| primer 2 || 1.0
+
| 10 μM primer 2 || 1.0
|-
|-
| 2x GoTaq || 25.0
| 2x GoTaq || 25.0
Line 55: Line 55:
| bgcolor=#cfcfcf | ng/μL  
| bgcolor=#cfcfcf | ng/μL  
|-
|-
-
| 1. H2B+his || 0.04 || 1.667 || 40.4
+
| 1. H2B || 0.04 || 1.667 || 40.4
|-
|-
-
| 2. LOV || 0.019 || 1.562 || 18.6
+
| 2. LOV+his || 0.019 || 1.562 || 18.6
|-
|-
| 3. pSB1A3 || 0.069 || 1.754 || 69.4
| 3. pSB1A3 || 0.069 || 1.754 || 69.4
Line 77: Line 77:
| pSB1A3 || 2000 || 85.5 || 12.2 || 27.8
| pSB1A3 || 2000 || 85.5 || 12.2 || 27.8
|-
|-
-
| H2B+his || 396 || 40.4 || 5.1 || 34.9
+
| H2B || 378 || 40.4 || 4.9 || 35.1
|-
|-
-
| LOV || 429 || 18.6 || 12.0 || 28.0
+
| LOV+his || 450 || 18.6 || 12.6 || 27.4
|}
|}
-
(02/24/13)<br>
+
(02/25/13)<br>
Golden Gate Reactions
Golden Gate Reactions
-
# LOV + H2B+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 || ---
+
| pSB1A3 gg || 1.0 || 1.0 || --- || 2.0 || 2.0 || ---
|-
|-
-
| gg3 LOV || 1.0 || H2O || ---
+
| H2B gg || 1.0 || H2O || --- || 2.0 || H2O || ---
|-
|-
-
| gg4 H2B+his || 1.0 || H2O || ---
+
| LOV+his gg || 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
+
* 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
* [45°C, 2 min.; 16°C, 5 min.] x25
* [45°C, 2 min.; 16°C, 5 min.] x25
-
* 60°C, 20 min.
+
* 60°C, 10 min.
* 80°C, 20 min.
* 80°C, 20 min.
* 4°C, ∞
* 4°C, ∞
 +
 +
Transformation
 +
* Transfer 10 μL reactions into 2.0 mL round bottom tubes; add 50 μL chemically competent BL21 cells (thawed on ice); pipette up and down 3x to mix
 +
* Incubate on ice for 5 min.
 +
* Heat shock at 42°C (heat block) for exactly 45 sec.; place on ice immediately
 +
* Add 750 μL plain LB broth; 37°C incubator: lay tubes flat and tape to the shaking rack; incubate with shaking for 45 min.
 +
* Pellet the cells at top speed for 3 min. at room temp.
 +
* Discard the supernatant; resuspend the pellet in 100 μL LB Amp (100μg/mL); plate on LB Amp agar (100 μg/mL)
 +
* Incubate overnight
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Current revision

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
10 μM primer 1 1.0
10 μM 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, 10 min.
  • 80°C, 20 min.
  • 4°C, ∞


Transformation

  • Transfer 10 μL reactions into 2.0 mL round bottom tubes; add 50 μL chemically competent BL21 cells (thawed on ice); pipette up and down 3x to mix
  • Incubate on ice for 5 min.
  • Heat shock at 42°C (heat block) for exactly 45 sec.; place on ice immediately
  • Add 750 μL plain LB broth; 37°C incubator: lay tubes flat and tape to the shaking rack; incubate with shaking for 45 min.
  • Pellet the cells at top speed for 3 min. at room temp.
  • Discard the supernatant; resuspend the pellet in 100 μL LB Amp (100μg/mL); plate on LB Amp agar (100 μg/mL)
  • Incubate overnight


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