User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/05

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(Autocreate 2013/01/05 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
Current revision (22:42, 5 January 2013) (view source)
(01/05/13)
 
(24 intermediate revisions not shown.)
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| colspan="2"|
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-
==mm/dd/yy==
+
==01/05/13==
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-
* Line item 1
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* Gibson: PCR parts, complete protocol for Brady's construct
-
* Line item 2
+
* Golden gate: PCR parts; waiting on BsmBI from NEB
----
----
-
'''Minipreps'''<br>
+
'''Gibson Assembly'''<br>
-
* Check with E/P digests
+
# hPCD + BL01 + V0120
 +
# hPCD + BL01 + pSB1A3
 +
 
 +
 
 +
'''PCR'''
 +
# V0120, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002)
 +
# pSB1A3, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002)
 +
# hPCD, BBP-hPCD fwd (BL9) / hPCD-BL01 rev (BL7)
 +
# BL01, hPCD-BL01 fwd (BL8) / BL01-BBS rev (BL10)
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
Line 20: Line 28:
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
-
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
+
| rowspan="7" | <u>Expected:</u><br>G1. V0120 = 3200<br>G2. pSB1A3 = 2000<br>G3. hPCD = 186<br>G4. BL01 = 2520
-
| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
+
| rowspan="7" | [[Image:KAH010513_gel1.jpg|300px|PCR gel]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
-
| DNA(plasmid) || 2.0 μL
+
| DNA(plasmid) || 0.5 μL
|-
|-
-
| 10X buffer || 1.5
+
| 10 μM primer 1 || 1.0
|-
|-
-
| EcoRI || 1.0
+
| 10 μM primer 2 || 1.0
|-
|-
-
| PstI || 1.0
+
| 2x GoTaq mix || 25.0
|-
|-
-
| dH<sub>2</sub>O || 9.5
+
| dH<sub>2</sub>O || 22.5
|-
|-
-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
+
| &nbsp; || 50 μL  
|}
|}
-
----
+
* 95°C, 3 min.
-
'''Assemblies'''
+
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
-
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
+
* 72°C, 3 min.
-
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
+
* 4°C, ∞
-
* Digests (Fermentas FD)
+
'''Purification'''
-
** Specific notes
+
* Cleaned remaining 40 μL of PCR using the Zymo DNA Clean & Concentrator kit
 +
* Eluted samples with 20 μL dH<sub>2</sub>O
-
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
+
 
 +
'''DNA Concentrations'''
 +
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
|- valign="top"
-
| bgcolor=#cfcfcf | Reagent
+
| bgcolor=#cfcfcf | Sample
-
| bgcolor=#cfcfcf | Volume
+
| bgcolor=#cfcfcf | OD 260
-
| rowspan="7" | <!-- [[Image:GelImage.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
+
| bgcolor=#cfcfcf | 260/ 280
-
|-
+
| bgcolor=#cfcfcf | ng/μL
-
| DNA (plasmid) || up to 25 μL
+
|-
|-
-
| 10x buffer || 3.0
+
| G1 V0120 || 0.108 || 1.801 || 108.375
|-
|-
-
| enzyme 1 || 1.0
+
| G2 pSB1A3 || 0.101 || 1.849 || 100.553
|-
|-
-
| enzyme 2 || 1.0
+
| G3 hPCD || 0.034 || 1.876 || 33.586
|-
|-
-
| dH<sub>2</sub>O || ---
+
| G4 BL01 || 0.113 || 1.828 || 112.79
-
|-
+
-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
+
|}
|}
-
* Measure conc.'s
+
'''Reaction set-up'''
-
{| {{table}} cellspacing="3" <!-- [DNA] table -->
+
* Use 0.2 - 0.5 pmol, per NEB's suggestion, [http://www.neb.com/nebecomm/products/protocol819.asp]
-
|- bgcolor=#cfcfcf
+
* pmols = ng x 1000 / bp x 650 daltons; ng = (pmols x bp x 650 daltons) / 1000
-
| Sample || OD260 || 260/280 || ng/μL
+
* Total volume of DNA = 5.0 μL max
-
|-
+
-
| 1. Digested part (a/b) || --- || --- || ---
+
-
|-
+
-
| 2. Digested part (c/d) || --- || --- || ---
+
-
|}
+
 +
1. hPCD + BL01 + V0120
 +
* vector V0120, 0.1 pmol = 208 ng = 1.9 μL (use 1.4 μL)
 +
* 2:1 insert hPCD, 0.2 pmol = 24 ng = 0.7 μL
 +
* 2:1 insert BL01, 0.2 pmol = 328 ng = 2.9 μL
-
* Dephosphorylation (Roche)
+
2. hPCD + BL01 + V0120
-
{| {{table}} cellspacing="3" <!-- Dephos table -->
+
* vector pSB1A3, 0.1 pmol = 130 ng = 1.3 μL
-
|-
+
* 2:1 insert hPCD, 0.2 pmol = 24 ng = 0.7 μL (use 0.8 μL)
 +
* 2:1 insert BL01, 0.2 pmol = 328 ng = 2.9 μL
 +
 
 +
 
 +
Gibson reactions:
 +
# hPCD + BL01 + V0120
 +
# V0120
 +
# hPCD + BL01 + pSB1A3
 +
# pSB1A3
 +
 
 +
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 +
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
-
| bgcolor=#cfcfcf | Volume
+
| 1 || 2 || 3 || 4
|-
|-
-
| DNA (clean digest) || up to 17 μL (500 ng)
+
| Vector || 1.4 || 1.4 || 1.3 || 1.3
|-
|-
-
| 10x buffer d.p. || 2.0
+
| Ins 1 || 0.7 || --- || 0.8 || ---
|-
|-
-
| phosphatase || 1.0
+
| Ins 2 || 2.9 || --- || 2.9 || ---
|-
|-
-
| dH<sub>2</sub>O || ---
+
| dH<sub>2</sub>O || --- || 3.6 || --- || 3.7
|-
|-
-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
+
| &nbsp; || 5.0 μL || 5.0 μL || 5.0 μL || 5.0 μL
|}
|}
 +
* Add each to one aliquot of 15.0 Gibson mix (Rene); total = 20.0 μL
 +
* Thermal cycler: 50°C,60 min.; 4°C, ∞
-
* Ligations
 
-
{| {{table}} cellspacing="3" <!-- Ligations table -->
 
-
|- bgcolor=#cfcfcf
 
-
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
 
-
|-
 
-
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
 
-
|-
 
-
| 2. vector(c/d)/ ## ng || &nbsp;
 
-
|}
 
-
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
+
'''Transformation'''
-
| &nbsp;            || 1    || 2    ||
+
* (1) Transform: add '''2.0 μL''' to 40 μL DH5α-Turbo; ice/5 min.; plate on warm 100 μg/mL amp. agar
 +
* (2) NEB also suggests diluting 5.0 μL of the Gibson reaction into 15.0 μL dH<sub>2</sub>O (20.0 total vol.), and using 2.0 μL diluted DNA, [http://www.neb.com/nebecomm/products/protocol820.asp]
 +
* Test both methods (8 plates total)
 +
** Plates 1-4, method 1
 +
** Plates 5-8, method 2
 +
 
 +
 
 +
----
 +
'''Golden Gate Assembly'''<br>
 +
* Try new protocol from Dave Savage
 +
# hPCD + BL01 + V0120
 +
# hPCD + BL01 + pSB1A3
 +
 
 +
 
 +
* Check the sequences for BsmBI docking sites (CGTCTC)
 +
** hPCD - none
 +
** BL01: hPCD-mCherry-SP1AB - none
 +
** V0120 - <font color="red">5 sites</font>
 +
** pSB1A3 - none
 +
 
 +
 
 +
'''PCR'''
 +
# V0120, gg_BBS F (gg0001) / gg_BBP R (gg0002)
 +
# pSB1A3, gg_BBS F (gg0001) / gg_BBP R (gg0002)
 +
# hPCD, gg_BBP-hPCD F (gg0003) / gg_hPCD-BL01 R (gg0004)
 +
# BL01, gg_BL01 F (gg0004) / gg_BL01-BBS R (gg0005)
 +
 
 +
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf | Volume
 +
| rowspan="7" | <u>Expected:</u><br>gg1. V0120 = 3200<br>gg2. pSB1A3 = 2000<br>gg3. hPCD = 186<br>gg4. BL01 = 2520
 +
| rowspan="7" | [[Image:KAH010513_gel1.jpg|300px|PCR gel]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
-
| Insert DNA         || ###  || ---  ||
+
| DNA(plasmid) || 0.5 μL
|-
|-
-
| Vector DNA        || ###  || ###  ||
+
| 10 μM primer 1 || 1.0
|-
|-
-
| 2x lgn buf (Roche) || ###  || ###  ||
+
| 10 μM primer 2 || 1.0
|-
|-
-
| T4 ligase (NEB)    || 1.0 || 1.0  ||
+
| 2x GoTaq mix || 25.0
|-
|-
-
| dH<sub>2</sub>O   || ###  || ###  ||
+
| dH<sub>2</sub>O || 22.5
|-
|-
-
| &nbsp;             || # μL || # μL ||
+
| &nbsp; || 50 μL  
|}
|}
-
----
+
* 95°C, 3 min.
-
'''Oligo annealing'''
+
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
-
# New BB 1
+
* 72°C, 3 min.
-
# New BB 2
+
* 4°C, ∞
-
{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
+
 
-
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
+
 
 +
'''Purification'''
 +
* Cleaned remaining 40 μL of PCR using the Zymo DNA Clean & Concentrator kit
 +
* Eluted samples with 20 μL dH<sub>2</sub>O
 +
 
 +
 
 +
'''DNA Concentrations'''
 +
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Sample
 +
| bgcolor=#cfcfcf | OD 260
 +
| bgcolor=#cfcfcf | 260/ 280
 +
| bgcolor=#cfcfcf | ng/μL
|-
|-
-
| 10x annealing buffer || 2.0
+
| gg1 V0120 || 0.099 || 1.834 || 98.549
|-
|-
-
| dH<sub>2</sub>O || ---
+
| gg2 pSB1A3 || 0.068 || 1.758 || 67.725
|-
|-
-
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
+
| gg3 hPCD || 0.022 || 1.72 || 22.485
 +
|-
 +
| gg4 BL01 || 0.056 || 1.707 || 55.883
|}
|}
 +
 +
 +

Current revision

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

01/05/13

  • Gibson: PCR parts, complete protocol for Brady's construct
  • Golden gate: PCR parts; waiting on BsmBI from NEB



Gibson Assembly

  1. hPCD + BL01 + V0120
  2. hPCD + BL01 + pSB1A3


PCR

  1. V0120, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002)
  2. pSB1A3, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002)
  3. hPCD, BBP-hPCD fwd (BL9) / hPCD-BL01 rev (BL7)
  4. BL01, hPCD-BL01 fwd (BL8) / BL01-BBS rev (BL10)
Reagent Volume Expected:
G1. V0120 = 3200
G2. pSB1A3 = 2000
G3. hPCD = 186
G4. BL01 = 2520
PCR gel
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.5 μL
10 μM primer 1 1.0
10 μM primer 2 1.0
2x GoTaq mix 25.0
dH2O 22.5
  50 μL
  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
  • 72°C, 3 min.
  • 4°C, ∞


Purification

  • Cleaned remaining 40 μL of PCR using the Zymo DNA Clean & Concentrator kit
  • Eluted samples with 20 μL dH2O


DNA Concentrations

Sample OD 260 260/ 280 ng/μL
G1 V0120 0.108 1.801 108.375
G2 pSB1A3 0.101 1.849 100.553
G3 hPCD 0.034 1.876 33.586
G4 BL01 0.113 1.828 112.79


Reaction set-up

  • Use 0.2 - 0.5 pmol, per NEB's suggestion, [1]
  • pmols = ng x 1000 / bp x 650 daltons; ng = (pmols x bp x 650 daltons) / 1000
  • Total volume of DNA = 5.0 μL max

1. hPCD + BL01 + V0120

  • vector V0120, 0.1 pmol = 208 ng = 1.9 μL (use 1.4 μL)
  • 2:1 insert hPCD, 0.2 pmol = 24 ng = 0.7 μL
  • 2:1 insert BL01, 0.2 pmol = 328 ng = 2.9 μL

2. hPCD + BL01 + V0120

  • vector pSB1A3, 0.1 pmol = 130 ng = 1.3 μL
  • 2:1 insert hPCD, 0.2 pmol = 24 ng = 0.7 μL (use 0.8 μL)
  • 2:1 insert BL01, 0.2 pmol = 328 ng = 2.9 μL


Gibson reactions:

  1. hPCD + BL01 + V0120
  2. V0120
  3. hPCD + BL01 + pSB1A3
  4. pSB1A3
Reagent 1 2 3 4
Vector 1.4 1.4 1.3 1.3
Ins 1 0.7 --- 0.8 ---
Ins 2 2.9 --- 2.9 ---
dH2O --- 3.6 --- 3.7
  5.0 μL 5.0 μL 5.0 μL 5.0 μL
  • Add each to one aliquot of 15.0 Gibson mix (Rene); total = 20.0 μL
  • Thermal cycler: 50°C,60 min.; 4°C, ∞


Transformation

  • (1) Transform: add 2.0 μL to 40 μL DH5α-Turbo; ice/5 min.; plate on warm 100 μg/mL amp. agar
  • (2) NEB also suggests diluting 5.0 μL of the Gibson reaction into 15.0 μL dH2O (20.0 total vol.), and using 2.0 μL diluted DNA, [2]
  • Test both methods (8 plates total)
    • Plates 1-4, method 1
    • Plates 5-8, method 2



Golden Gate Assembly

  • Try new protocol from Dave Savage
  1. hPCD + BL01 + V0120
  2. hPCD + BL01 + pSB1A3


  • Check the sequences for BsmBI docking sites (CGTCTC)
    • hPCD - none
    • BL01: hPCD-mCherry-SP1AB - none
    • V0120 - 5 sites
    • pSB1A3 - none


PCR

  1. V0120, gg_BBS F (gg0001) / gg_BBP R (gg0002)
  2. pSB1A3, gg_BBS F (gg0001) / gg_BBP R (gg0002)
  3. hPCD, gg_BBP-hPCD F (gg0003) / gg_hPCD-BL01 R (gg0004)
  4. BL01, gg_BL01 F (gg0004) / gg_BL01-BBS R (gg0005)
Reagent Volume Expected:
gg1. V0120 = 3200
gg2. pSB1A3 = 2000
gg3. hPCD = 186
gg4. BL01 = 2520
PCR gel
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.5 μL
10 μM primer 1 1.0
10 μM primer 2 1.0
2x GoTaq mix 25.0
dH2O 22.5
  50 μL
  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
  • 72°C, 3 min.
  • 4°C, ∞


Purification

  • Cleaned remaining 40 μL of PCR using the Zymo DNA Clean & Concentrator kit
  • Eluted samples with 20 μL dH2O


DNA Concentrations

Sample OD 260 260/ 280 ng/μL
gg1 V0120 0.099 1.834 98.549
gg2 pSB1A3 0.068 1.758 67.725
gg3 hPCD 0.022 1.72 22.485
gg4 BL01 0.056 1.707 55.883




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