Wed, Aug 04 21

pFC PCR and pFC9 digest: Round 3 #

pFC9 EcoRI SacI digest #

Yesterday I digested three 1ug samples of pFC9 with SacI and EcoRI to produce the large fragment needed for T7 initiation series assemblies.

Sample 1 #

First gel ran was standard 0.08 TAE at 120 V for 1 hr. Ran sample 1 in four separate lanes because I poured very thin gels and was bit worried about overtopping the wells.

Forth well was a bit lower than the other three due to bent comb, it did not actually run faster.

I then extracted all large fragment bands using UV table and tin foil method. Added all bands to the same tube and extracted using Zymoclean protocol.

Sample 2 #

Ran another gel in order to attempt the gel extraction again.

However this time I eluted DNA from column with 30 ul water instead of the elution buffer.

Nanodrop results #

Sample NameSample numberng/ul260/280260/230
pFC9 EcoRI SacI digest116.61.449-0.147
pFC9 EcoRI SacI digest216.11.677-0.307

Not great, looks basically exactly like previous extractions. At least I am consistent!


Did the same PCR, exact same protocol, DNA and reagents from yesterday. Only difference was I did two replicates of each sample. Ran “A” samples out on gel and put “B” samples in VR-inserts box.

As usual gel looked good with high signal from expected bands at ~300 bp. I then extracted each band on the gel UV table using tin foil to protect bands. Then extracted DNA from the agarose following Zymogen protocol and eluted DNA with 30ul water.

Nanodrop results #

Sample nameSample numberng/ul260/280260/230
pFC8tac (EH)112.21.820-0.255
pFC9 (EH)23.02.010-0.038
pFC8 (EH)31.23.7-0.01
pFC8T1T2 (RL)41.21.706-0.018
pFC8 (RL)5-0.10.1510.002
pFC53(8)T1T2 (RL)62.21.823-0.037
pFC8T2 (RL)72.13.463-0.047
pFC8tac (EH)816.11.677-0.307

Overall worse then yesterday. This may mean that using the elution buffer is better than H20, however there are too many untested variables at this point to make that determination especially considering that eluting with H20 did not improve the pFC9 results.

Overall all assays from the last three days have shown generally low yields and aparent contaimination by protein (260/280) and salt (260/230). Lower values are worse.

Plots generated from this Jupyter notebook.