User:Karmella Haynes/Notebook/BioBrick cloning/2010/08/03

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

08/03/10

  • ✓ Assemblies: Pc-ATF's and Pc-RFP's + Kozak
  • ✓ Minipreps: KFV1 (2)



Minipreps
> Check with separate NotI and SpeI digests

Reagent Volume Expected:
1, 3. KFV1/NotI = ~7000(+~35)
2, 4. KFV1/SpeI = ~7000(+~35)
Digest 8/03/10
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
enzyme 2.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

--> Keep clone #1; figure out what primer to use for sequencing confirmation (Ian's Actin 5C sequence data)


Assemblies

  1. KAH160: Kozak(K0001)/(S/P)/~3200 ✓ + (1) KAH109/(X/P)/1123
  2. KAH161: " + (2) KAH111/(X/P)/1123
  3. KAH162: " + (3) KAH112/(X/P)/1123
  4. KAH163: " + (4) KAH113/(X/P)/2107
  5. KAH164: " + (5) KAH114/(X/P)/2110
  6. KAH165: " + (6) KAH148/(X/P)/967
  7. KAH166: " + (7) KAH150/(X/P)/967
  8. KAH167: " + (8) KAH151/(X/P)/967
  9. KAH168: " + (9) KAH152/(X/P)/1931
  10. KAH169: " + (10) KAH1153/(X/P)/1934
  11. KAH170: " + (11) KAH115/(X/P)/931


> Digests (Fermentas FD)

Reagent Volume   Cloning digest 8/03/10
30 μL/lane, 1% agarose
DNA (plasmid) 15.0
10x buffer 3.0
XbaI 1.0
PstI 1.0
dH2O 10.0
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH109 (X/P) 1.057 2.43 52.8
2. KAH111 (X/P) 0.827 2.21 41.4
3. KAH112 (X/P) 1.089 2.53 54.4
4. KAH113 (X/P) 1.380 1.94 69.0
5. KAH114 (X/P) 1.022 2.20 51.1
6. KAH148 (X/P) 0.793 2.25 39.6
7. KAH150 (X/P) 0.660 2.09 33.0
8. KAH151 (X/P) 0.631 2.08 31.6
9. KAH152 (X/P) 1.030 2.12 51.5
10. KAH153 (X/P) 0.980 2.14 49.0
11. KAH115 (X/P) 0.628 2.21 31.4


> Ligations

Ligation Plate results (lig : neg crtl) 08/04/10
1. KAH109(X/P)/1123, 18 ng + K0001(S/P)/3200, 25 ng KAH160 >10:1 (Pick 2)
2. KAH111(X/P)/1123, 18 ng + " KAH161 >10:1 (Pick 2)
3. KAH112(X/P)/1123, 18 ng + " KAH162 >10:1 (Pick 2)
4. KAH113(X/P)/2107, 33 ng + " KAH163 >10:1 (Pick 2)
5. KAH114(X/P)/2110, 33 ng + " KAH164 >10:1 (Pick 2)
6. KAH148(X/P)/967, 15 ng + " KAH165 >10:1 (Pick 2)
7. KAH150(X/P)/967, 15 ng + " KAH166 >10:1 (Pick 2)
8. KAH151(X/P)/967, 15 ng + " KAH167 >10:1 (Pick 2)
9. KAH152(X/P)/1931, 30 ng + " KAH168 >10:1 (Pick 2)
10. KAH153(X/P)/1934, 30 ng + " KAH169 >10:1 (Pick 2)
11. KAH115(X/P)/931, 15 ng + " KAH170 >10:1 (Pick 2)
12. K0001(S/P)/ 25 ng 0 colonies
  1-11 12
Insert DNA 1.0 ---
Vector DNA 0.4 0.4
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 2.6 2.6
  10μL 10μL



Personal tools