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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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 DTT1
10mM ATP1
FD BsaI1
T4 DNA ligase0.5
M.B. H2O0
Total25.4

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 mix25
MB grade H2O0
DBN003 f1 primer0.25
DBN002 r1 primer0.25
template24.5
Total50
Step Temperature °C Duration
Initial denature95120 sec
Main Cycle (x40):
denature9530 sec
anneal5930 sec
extension72120 sec
final extension72300 sec
soak4indef.

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)
DNA2515
10x Green FD Buffer33
EcoRI11
SpeI01
XbaI10
Water010
Total3030

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

Gel image:

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
KAH575.1281.4580541832.2275
KAH1047.93050.9072182.451

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)
vector10
insert1.5
Roche Buffer 2x10
NEB T4 ligase2
Total23.5

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)


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