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

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* 4°C, ∞
* 4°C, ∞
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{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
+
{| {{table}} border="1" cellspacing="3" <!-- PCR rxn. table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
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Dilutions: to final concentration of 20 fmol/μL (40 μL)
Dilutions: to final concentration of 20 fmol/μL (40 μL)
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{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
+
{| {{table}} border="1" cellspacing="3" <!-- Dilution table: x = length in bp ÷ measured ng/μL * 0.26 * 2 -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | DNA
| bgcolor=#cfcfcf | DNA
| bgcolor=#cfcfcf | length (bp)
| bgcolor=#cfcfcf | length (bp)
| bgcolor=#cfcfcf | ng/μL
| bgcolor=#cfcfcf | ng/μL
-
| bgcolor=#cfcfcf | Vol.
+
| bgcolor=#cfcfcf | Vol. (x)
| bgcolor=#cfcfcf | + dH<sub>2</sub>O
| bgcolor=#cfcfcf | + dH<sub>2</sub>O
|-
|-
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| pSB1A3 || 2000 || 85.5 || --- || ---
+
| pSB1A3 || 2000 || 85.5 || 12.2 || 27.8
|-
|-
-
| H2B+his || 396 || 40.4 || --- || ---
+
| H2B+his || 396 || 40.4 || 5.1 || 34.9
|-
|-
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| LOV || 429 || 66.9 || --- || ---
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| LOV || 429 || 18.6 || 12.0 || 28.0
|}
|}
Golden Gate Reactions
Golden Gate Reactions
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# hPCD + BL01 + pSB1A3
+
# LOV + H2B+his + pSB1A3
# pSB1A3
# pSB1A3
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| gg4 BL01 || 1.0 || ---  
| gg4 BL01 || 1.0 || ---  
|-
|-
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| 10x ligase buffer || 1.0 || 1.0
+
| 10x PRO ligase buffer || 1.0 || 1.0
|-
|-
| NEB T4 lgase || 0.25 || 0.25
| NEB T4 lgase || 0.25 || 0.25

Revision as of 21:21, 23 February 2013

Karmella's BioBrick Cloning Main project page
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02/23/13

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


Type IIS Assembly, PCR: LOV-H2B+his

  1. H2B = 378 bp, ggH2B f/r (Template: H2B-GFP from Addgene); note: reverse primer adds a 6-his tag (18 bp)
  2. LOV = 429 bp, ggLOV f/r (Template: LOV from Addgene)
  3. pSB1A3 = ~2000, gg0001/0002 v2

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 = 396
2. LOV = 429
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)

DNA length (bp) ng/μL Vol. (x) + dH2O
pSB1A3 2000 85.5 12.2 27.8
H2B+his 396 40.4 5.1 34.9
LOV 429 18.6 12.0 28.0


Golden Gate Reactions

  1. LOV + H2B+his + pSB1A3
  2. pSB1A3
Reagent 1 2
gg2 pSB1A3 1.0 1.0
gg3 hPCD 1.0 ---
gg4 BL01 1.0 ---
10x PRO ligase buffer 1.0 1.0
NEB T4 lgase 0.25 0.25
BsmBI 0.5 0.5
dH2O 5.25 7.25
  10.0 10.0

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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