Mon, Jul 26 21

Confirming midi prep results #

Today I am working on visualizing the results of the midi preps I did for pFC8, 9 and 53tac (see notes from 7-20-21 to 7-23-21).

KpnI + HindIII double digest #

First I utilized a restriction digest with HindIII and KpnI to confirm plasmid ids and check for contaminating DNAs. Link to spreadsheet used for reagent calculation can be found here.

Reagents #

ReagentExpirationLot number
Buffer 2.15/2310070034

KpnI was expired but it did not seem to significantly effect results.

Results #

I expected to see results similar to the simulated gel shown below.

MW:  1 kb DNA Ladder

1:  pFC8
    HindIII + KpnI
       1. 2608 bp
       2. 981 bp

2:  pFC9
    HindIII + KpnI
       1. 2616 bp
       2. 973 bp

3:  pFC53tacT1T2
    HindIII + KpnI
       1. 2935 bp
       2. 1385 bb

I digested each plasmid for 30 mins at 37C and then ran them on a 0.8% agarose TAE gel for 1 hr at 90 volts.

Overall both pFC8 and pFC9 look as expect although they cannot be distinguished with REs. pFC54tac does show the shift upwards on the large fragment but in a less dramatic fashion as the simulated gel. This is even more true for the smaller (expected 1385 bp) fragment which shows little differentiation from the ~980 bp fragments produced from pFC8 and pFC9.

BamHI-HF + KpnI-HF double digest #

After running the KpnI HindIII digest and looking back at the plasmid maps I realized there was a much better digest that I could have done that would in theory resolve all three plasmids from each other. Digesting with BamHI and KpnI takes advantage of the reversed position KpnI site on pFC8 and 9 and using BamHI which is located downstream of the T1T2 terminators on pFC53tac (which pFC8 and 9 lack) means all three plasmids will produce unique fragments.

So I repeated by protocol but used BamHI-HF and KpnI-HF enzymes with CutSmart buffer. I used HF version because these cutters normally do not have good activity together.

Reagents #

ReagentExpirationLot number
CutSmart Buffer9/2210046090

Results #

Here both pFC8 and pFC9 look like pFC8 in the simulated gel. This makes me think that I accidentally added pFC8 to the pFC9 tube of the transformation.

I think overall the results for pFC53tac conform to expectations, simulated gel for the two digests done below.

MW:  1 kb DNA Ladder

1:  pFC53tacT1T2
    KpnI + BamHI
       1. 2557 bp
       2. 1763 bp

2:  pFC53tacT1T2
    KpnI + HindIII
       1. 2935 bp
       2. 1385 bp

Tomorrow I will re-run this gel but using Fred’s pFC8 and pFC9 (the same plasmid used in notes from 7-20-21) and start transformation with pFC9 again because I am thinking that is the most likely explanation.