User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/16: Difference between revisions

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|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1,2
| bgcolor=#cfcfcf | Rxn1,3
| bgcolor=#cfcfcf | Rxn3,4
| bgcolor=#cfcfcf | Rxn2,4
| rowspan="7" | Expected:<br>1,2. Gal4DB-mCh = 1170<br>3,4. ATF2 = 906<br>
| rowspan="7" | Expected:<br>MV10 band = 5191
| rowspan="7" | [[Image:KAH041615_gel1.jpg|250px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan="7" | [[Image:KAH041615_gel1.jpg|250px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
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* 72°C, 10 min
* 72°C, 10 min
* 4°C ∞
* 4°C ∞
Conclusion
* Not ideal, proceed with caution
Measure conc.
* PCR 1 & 2 combined, purified with Sigma PCR clean-up kit
* Eluted& back-eluted with 30 μL elution sln.
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
| 1. 5'p-MV10 PCR || 0.008 || 1.28 || 7.77
|}




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LabNet OptiMax Thermocycler (AnOlig RD)
LabNet OptiMax Thermocycler: AnOlig RD
* 37°C, 30 min
* 37°C, 30 min
* 95°C, 5 min
* 95°C, 5 min
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Digestion/ Ligation Reactions (Vector/ dsOligo Insert)
Digestion/ Ligation Reactions (Vector / dsOligo Insert)
# ###/###
# pX330 / g051
# ###/###
# pX330 / g052
# ###/###
# pX330 / g053
# pX330 / g054
# pX330 / g055
# pX330 / g056




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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn
| bgcolor=#cfcfcf | Rxn
| bgcolor=#cfcfcf | Mix (x7)
|-
|-
| 100 ng Vector || ##
| 100 ng Vector || 0.5 || 3.5
|-
|-
| 1:250 dsOligo || 2.0  
| 1:250 dsOligo || 2.0 || ---
|-
|-
| 10x FD buf (NEB) || 2.0  
| 10x FD buf (NEB) || 2.0 || 14.0
|-
|-
| 10 mM DTT || 1.0
| 10 mM DTT || 1.0 || 7.0
|-
|-
| 10 mM ATP || 1.0
| 10 mM ATP || 1.0 || 7.0
|-
|-
| FastDigest BbsI || 1.0
| FastDigest BbsI || 1.0 || 7.0
|-
|-
| T7 DNA ligase || 0.5  
| T7 DNA ligase || 0.5 || 3.5
|-
|-
| dH<sub>2</sub>O || ##
| dH<sub>2</sub>O || 12.0 || 84.0
|-
|-
| &nbsp; || 20.0
| &nbsp; || 20.0
|}
|}


Bio-Rad Thermocycler
LabNet OptiMax Thermocycler: BbsI Dig/Lig
* 6x [37°C, 5 min; 23°C, 5 min]
* 6x [37°C, 5 min; 23°C, 5 min]
* 4°C, ∞
* 4°C, ∞
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Transformation(s)
Transformation(s)
* 2.0 μL ligation + 50 μL DH5α-turbo
* 10.0 μL ligation + 50 μL DH5α-turbo
* Plate on 100 μg/mL amp  
* Plate on 100 μg/mL amp  
** Split ligations in 1/2, do rapid protocl for 1 set, long protocol for other set




Revision as of 17:49, 16 April 2015

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04/16/15

  • LCR Development - PCR MV10 bacbone
  • gRNA cloning (Rene)


LCR Development

  • Try PCR on linearized vector
Reagent Volume
DNA(plasmid) 5.0 μL
10X buffer 3.0
XbaI 2.0
dH2O 20.0
  30 μL --> 37°C/ ~30 min.


Measure conc.

  • DNA purified with Sigma PCR clean-up kit
  • Eluted& back-eluted with 30 μL elution sln.
Sample OD260 260/280 ng/μL
1. MV10 XbaI 0.017 1.47 17.24


Phusion PCR

  1. 1.0 μL template, HF buffer
  2. 2.0 μL template, HF buffer
  3. 1.0 μL template, GC buffer
  4. 2.0 μL template, GC buffer
Reagent Rxn1,3 Rxn2,4 Expected:
MV10 band = 5191 		Hover name
10 μL/lane, 1% agarose; Ladder
Template 1.0 2.0
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 (HF/GC) 5.0 5.0
dH2O 40.5 39.5
  50.0 50.0

Program: Phusion (block B)

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

Conclusion

  • Not ideal, proceed with caution


Measure conc.

  • PCR 1 & 2 combined, purified with Sigma PCR clean-up kit
  • Eluted& back-eluted with 30 μL elution sln.
Sample OD260 260/280 ng/μL
1. 5'p-MV10 PCR 0.008 1.28 7.77


gRNA cloning - Rene

  • Follow the "Oligo annealing and cloning into backbone vectors - NEW" protocol

Phosphorylate and anneal oligo pairs

  1. g051 T/B
  2. g052 T/B
  3. g053 T/B
  4. g054 T/B
  5. g055 T/B
  6. g056 T/B
Reagent Rxn Mix (7x)
100 μM Oligo 1 1.0 ---
100 μM Oligo 2 1.0 ---
10x T4 Lign buf (NEB) 1.0 7.0
T4 PNK (NEB) 0.5 3.5
dH2O 6.5 45.5
  10.0


LabNet OptiMax Thermocycler: AnOlig RD

  • 37°C, 30 min
  • 95°C, 5 min
  • Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min]
  • 25°C, ∞


Dilute the product(s) 1:250

  • Add 2 μL product to 498 μL dH2O


Digestion/ Ligation Reactions (Vector / dsOligo Insert)

  1. pX330 / g051
  2. pX330 / g052
  3. pX330 / g053
  4. pX330 / g054
  5. pX330 / g055
  6. pX330 / g056


Reagent Rxn Mix (x7)
100 ng Vector 0.5 3.5
1:250 dsOligo 2.0 ---
10x FD buf (NEB) 2.0 14.0
10 mM DTT 1.0 7.0
10 mM ATP 1.0 7.0
FastDigest BbsI 1.0 7.0
T7 DNA ligase 0.5 3.5
dH2O 12.0 84.0
  20.0

LabNet OptiMax Thermocycler: BbsI Dig/Lig

  • 6x [37°C, 5 min; 23°C, 5 min]
  • 4°C, ∞


Transformation(s)

  • 10.0 μL ligation + 50 μL DH5α-turbo
  • Plate on 100 μg/mL amp
    • Split ligations in 1/2, do rapid protocl for 1 set, long protocol for other set




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