Team:Paris Bettencourt/Notebook/Binding
Week 27th June - 3rd July
Week 4th - 10th July
Week 11th - 17th July
NEB Ph.D.™-7 Phage Display Peptide Library Kit solutions preparation
NEB protocol recommends to prepare a mixed solution of ITPG and X-gal. Because I already have IPTG stock, I prefer to do 2 separate solution for IPTG and X-gal (also avoids throwing both IPTG and X-gal if anything were to happen to the tubes).
IPTG
M.W.= 238.31g/mol
NEB protocol recommends to put 1.25g in 25mL DMF, which is [IPTG]=50mg/mL
[IPTG]mass=[IPTG]/MW=0.05/238.31=0.21mol/L=210mM.
They recommend diluting 1000-fold to prepare plates. So [IPTG]working_concentration=50µg/mL which is 210µM.
In order to simplify things, I'll use [IPTG]working_concentration=200µM.
IPTG stocks are at 1M and 0.1M (100mM).
If preparing 1M stock, add 2.383g of IPTG to 10mL of milli-Q water. For 0.1M, add 238.3mg of IPTG to 10mL of milli-Q water.
2µL IPTG / mL medium should be added if using the 0.1M solution (0.2µL / mL of using the 1M solution).
X-gal
M.W.= 408.63g/mol
NEB protocol recommends to put 1g in 25mL DMF, which is [X-gal]=40mg/mL.
Sigma-Aldrich recommends having stock solution at 20mg/mL.
I decided to do a stock at 20mg/mL.
2 µL X-gal / mL of medium should be added.
Tetracycline
NEB protocol recommends doing a 20mg/mL stock solution in 50% EtOH. Most of the protocol found online recommend 70% EtOH, so that's what I did (it probably does not matter much).
300mg were dissolved in 15mL 70% EtOH.
TBS
NEB protocol recommends 50mM Tris-HCl (pH7.5), 150mM NaCl.
Tris-HCl 1M, pH 7.5:
M.W. Tris-Base=121.14g/mol
12.11g of Tris-Base were dissolved in 60mL of H2O.
pH was adjusted to 7.5 using HCl 5M.
Volume was adjusted to 100mL.
25 mL of Tris-HCl 1M, pH 7.5 were diluted in 500mL H2O.
M.W. NaCl=58.44g/mol
mNaCl=(58.44/1000)*150=8.766g for 1L
So 8.766/2=4.383g were added to the solution of Tris-HCl 50mM.
pH was measured after completion of the solution and was around 7.7.
Blocking Buffer
M.W. NaHCO3=84.007g/mol
2.1g of NaHCO3 were put in 225mL osmosed H2O.
pH was adjusted to 8.6 with 5M HCl.
Volume was adjusted to 250mL.
1.25 BSA was added to final concentration 5mg/mL.
Solution was kept 6 days at 4°C.
On 21/07/16, 0.05g NaN3 (0.02%) were added to the solution to avoid growth of microorganisms.
Solution was filter sterilized and aliquoted by ~75mL.
Week 18th -24th July
Week 25th -31th July
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