Randomized fragment

I am back from vacation and what a better way to shake my mind than a blog post!

Before my vacation break, I was trying to make a double stranded 200 bp fragment with a randomized region by using two different long oligos. One 135 bp with a 70 randomized bases and a 30 bases overlapping region with the other 95 bases oligo.

The 200 bp fragment is intended to make by annealing the two oligos and then extending/amplifying them by a modified PCR protocol.

In my last post, I described all the different strategies that I did in order to obtain that fragment. However, despite my several attempts, I always obtained I 250 bp product, instead of the 200 bp expected product.

new post

 

After analyzing the oligos sequence in detail, I realized that a un-specific annealing between regions of the oligos could be responsible of the extra 50 bases.

In order to test if this misspriming event was responsible of the extra bases, I sequenced the purified 250 fragment using both left and right primers.

Analysis of the sequence traces from the forward primer and from the reverse primer showed no evidence of misspriming at all.

REV.png

 

 

 

for2.png

 

In fact the sequenced fragment coincide with the expected 200 base pair fragment sequence and not to the product expected if this misspriming event had taken place.

In conclusion, I have no idea why the synthesized fragment migrated as a 250 bp fragment in the agarose gel electrophoresis.   However, I was able to show that the misspriming event did not happen. Two possibilities come to my mind to explain this problem:

2 pos.png

 

It is possible that some extra bases were added at the 3’ end of the fragment. This extra bases might not be detected in the sequenced fragment since the sequence trace lost resolution near the end of the fragment. However, even if this is the case the extra bases are not going to have negative consequences in the illumina barcoding attachment protocol step.

In this step, a set of primers containing a barcode and illumina flow-cell adapter sequences will anneal with both ends of the fragment. Then, the fragments will be amplified using a short cycle PCR protocol. Since the sequencing results show that the priming sites of this barcoding oligos are intact, then the extra bases won’t be expected to have any effect.

flow

Since no evidence of misspriming was detected, I consider that is safe to try some uptake experiments using the synthesized 250 base pair fragment and then quantify how much I can recover from the wild type Acinetobacter baylyi BD413 and from the no-uptake ADP239 comP::nptII mutant.

 

 

 

 

 

 

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