User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/20

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01/20/11

  • ✓ Cultures & minipreps: KAH179, 180, KAH176-178/MV8 (2 each); 5 mL ea./ 6 hrs.
  • ✓ pSB31 vector: for Pc-TF ES cell expression; diagnostic digest & retransformation



Minipreps
--> KAH179, 180: E/P/SacI
--> KAH176-178/MV8 fwd: E/ApaLI (*mCherry has an ApaLI site)
--> KAH176-178/MV8 rev: E/S

Reagent Volume Expected:
V0120 bands = 1785, 1440
1,2. KAH179 = 2941***
3,4. KAH180 = 2743

MV8 bands = 1243, 1135, 288, 234
**Last frag. of insert + 1655...
5,6. KAH176/MV8 fwd = 1609*, 711*, 2579**
7,8. KAH177/MV8 fwd = 1609*, 711*, 2772**
9,10. KAH178/MV8 = 1609*, 711*, 2783**

MV8 = 4594
11,12. KAH176/MV8 rev = 3191
13,14. KAH177/MV8 rev = 3384
KAH178/MV8 rev = 3395
File:KAH 012011 gel2.tif
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

--> Note: ***5xGal4 contains ScaI site, thus observed band size should be 2221 bp.



pBS31 vector
--> Collab. w/ Manching Ku (Bernstein lab); Use pBS31-RBGpA vector for FRT integration and dox-induced expression of Pc-TF in ES cells
--> Vector contains EcoR1 cloning site and NotI site elsewhere
--> Do diagnostic digest to check for XbaI, SpeI

Reagent Volume Expected:
1. EcoRI = 5127
2. XbaI = ?
3. SpeI = ?
4. no enzyme = uncut
File:KAH 012011 gel1.tif
15 μL/lane; 1% agarose
DNA(plasmid) 1.0 μL
10X buffer 1.5
enzyme 1.0
dH2O 11.5
  15 μL --> 37°C/ ~10 min.


--> Conclusion: Cannot use XbaI or SpeI for cloning in this vector. Blunt the vector and the insert.