User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/29: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 56: Line 56:
----
----
'''KAH126 expression - rebuild PcTF expression plasmid for K562'''
'''KAH126 expression - rebuild PcTF expression plasmid for K562'''
* Insert ### into MV10 (X)
* Insert KAH87 (X/S) into MV10 (X)
* KAH87 = hPCD : [mCh : VP64]


'''Minipreps'''<br>
* Check with E/P digests


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| 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] -->
|-
| DNA(plasmid) || 2.0 μL
|-
| 10X buffer || 1.5
|-
| EcoRI || 1.0
|-
| PstI || 1.0
|-
| dH<sub>2</sub>O || 9.5
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
|}
----
'''Assemblies'''
'''Assemblies'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
# KAH87/MV10: KAH87/(X/S)/1053 + MV10/(X)/5197
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size




* Digests (Fermentas FD)
* Digests (Fermentas FD)
** Specific notes
# KAH87
# MV10


{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Rxn 1
| bgcolor=#cfcfcf | Rxn 2
| 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] -->
| 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] -->
|-
|-
| DNA (plasmid) || up to 25 μL
| DNA (plasmid) || 15.0 || 15.0
|-
|-
| 10x buffer || 3.0
| 10x buffer || 3.0 || 3.0
|-
|-
| enzyme 1 || 1.0
| enzyme 1 || 1.0 || 1.0
|-
|-
| enzyme 2 || 1.0
| enzyme 2 || 1.0 || ---
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || 10.0 || 11.0
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| &nbsp; || 30.0 μL || 30.0 μL
|}
|}
--> 37°C/ ~30 min.
* Gel purify w/ Sigma kit
** Elute & back elute w/ 30 μL elution solution




Line 115: Line 99:
| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| 1. KAH87 (X/S) || --- || --- || ---
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| 2. MV10 (c/d) || --- || --- || ---
|}
|}


Revision as of 16:38, 29 April 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

04/29/15

  • LCR - Phusion PCR for MV10
  • KAH126 expression - rebuild PcTF expression plasmid for K562


MV10 Phusion PCR

  • Use linearized MV10 (XbaI-cut)
Reagent Rxn1 Rxn2 Expected:
1,2. MV10 = 5191 		Hover name
10 μL/lane, 1% agarose; Ladder
Template 0.5 0.5
10 uM fwd primer 1.0 1.0
10 uM rev primer 1.0 1.0
10 mM dNTPs 1.0 1.0
Phusion pol. 0.5 0.5
5x buffer 10.0 HF 10.0 GC
DMSO --- 1.5
dH2O 36.0 34.5
  50.0 50.0

Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • tba


KAH126 expression - rebuild PcTF expression plasmid for K562

  • Insert KAH87 (X/S) into MV10 (X)
  • KAH87 = hPCD : [mCh : VP64]


Assemblies

  1. KAH87/MV10: KAH87/(X/S)/1053 + MV10/(X)/5197


  • Digests (Fermentas FD)
  1. KAH87
  2. MV10
Reagent Rxn 1 Rxn 2
DNA (plasmid) 15.0 15.0
10x buffer 3.0 3.0
enzyme 1 1.0 1.0
enzyme 2 1.0 ---
dH2O 10.0 11.0
  30.0 μL 30.0 μL

--> 37°C/ ~30 min.


  • Gel purify w/ Sigma kit
    • Elute & back elute w/ 30 μL elution solution


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. KAH87 (X/S) --- --- ---
2. MV10 (c/d) --- --- ---


  • Dephosphorylation (Roche)
Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
Ligation Plate results (lig : neg crtl) mm/dd/yy
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #)
2. vector(c/d)/ ## ng  
  1 2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL

Oligo annealing

  1. New BB 1
  2. New BB 2
DNA (oligos, 100 μM) up to 18 μL (3 μL ea.)
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/04/29&oldid=882820"