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

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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).

Gel image:

2015-04-30 KAH57 & KAH104 digests annotated.png

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
KAH57 5.128 1.458054183 2.2275
KAH104 7.9305 0.907218 2.451

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)
vector 10
insert 1.5
Roche Buffer 2x 10
NEB T4 ligase 2
Total 23.5

Incubate for 10 minutes at room temperature.

Transformation

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)