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

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Ligation/Amplification of DBN002 & DBN003 Main project page
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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 DTT1
10mM ATP1
FD BsaI1
T4 DNA ligase0.5
M.B. H2O0

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.


Reagent Volume (uL)
2x master mix25
MB grade H2O0
DBN003 f1 primer0.25
DBN002 r1 primer0.25
Step Temperature °C Duration
Initial denature95120 sec
Main Cycle (x40):
denature9530 sec
anneal5930 sec
extension72120 sec
final extension72300 sec

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)
10x Green FD Buffer33

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

Gel image:


Cut out vector (0.134g) and insert (0.140g). Ran a gel extraction using Sigma GenElute Gel Extraction Kit, then measured concentration on the spectrophotometer.

Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280

Purity as measured by A260/A280 is poor, and concentration is low. Proceeding with ligation step.

50ng vector * 1/5.1 ng/µL = 10 µL purified KAH57 ng insert = 540bp/4691bp * 2 * 50ng = 11.5 ng, * 1/7.9 ng/µL = 1.5 µL purified KAH104

Reagent Volume (µL)
Roche Buffer 2x10
NEB T4 ligase2

Incubate for 10 minutes at room temperature.


Transform DH5α-Turbo with:

  • ✓ KAH57_v0120
  • ✓ KAH104_v0120
  • ✓ pMax-GFP (brochure says it needs a Kanamycin plate, but René says she used Amp, so try that first)
  • ✓ ligation product of KAH57 & KAH104
  • ✓ negative control (water)
  • ✓ negative control (digested KAH57 only)

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