Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/30

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Ligation/Amplification of DBN002 & DBN003 <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Overview

Going to try Type IIS Digestion/Ligation of DBN002 & DBN003, followed by PCR amplification. Also going to try conventional assembly using the BioBricks method .

Type IIS Digestion/Ligation

Reagent Volume (µL)
Insert 1 (DBN002) 200 ng (11.9 µL)
Insert 2 (DBN003) 500 ng (7.5 µL)
10x FD Buffer (clear) 2.5
10mM DTT 1
10mM ATP 1
FD BsaI 1
T4 DNA ligase 0.5
M.B. H2O 0
Total 25.4

Incubate the ligation reaction in a thermocycler (program name DBN IIS):

  1. 37°C 5 min
  2. 23°C 5 min
  3. Cycle the previous two steps for 6 cycles (total run time 1h)
  4. 4°C hold until ready to proceed

--Clean-up of IIS Ligation--

Use the Qiagen kit. If possible, final volume elution of 30 µL.

--PCR--

Reagent Volume (uL)
2x master mix 25
MB grade H2O 0
DBN003 f1 primer 0.25
DBN002 r1 primer 0.25
template 24.5
Total 50
Step Temperature °C Duration
Initial denature 95 120 sec
Main Cycle (x40):
denature 95 30 sec
anneal 59 30 sec
extension 72 120 sec
final extension 72 300 sec
soak 4 indef.

BioBricks Cloning

Follow the method described here.

Parts to be used are KAH57 (AmCyan, AmCyan, NLS, Stop) and KAH104 (HPK, Kozak). Both are in v0120 vector. For this cloning, KAH57 will be the vector and KAH104 the insert, placed in front of the KAH57 part. Enzymes used will be E/X for KAH57 and E/S for KAH104.

Reagent Volume (KAH57) Volume (KAH104)
DNA 25 15
10x Green FD Buffer 3 3
EcoRI 1 1
SpeI 0 1
XbaI 1 0
Water 0 10
Total 30 30

During gel extraction, remove the vector from KAH57 (should be only large piece in digest) & the insert from KAH104 (540bp).

Transformation

Transform DH5α-Turbo with:

  • KAH57
  • KAH104
  • pMax-GFP
  • ligation product of KAH57 & KAH104
  • ligation product of DBN002 & DBN003


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